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publication dateMar 30, 2015 publication descriptionACS Appl Mater Interfaces. 2015 Mar 30. [Epub ahead of print]ACS Applied Materials & Interfaces (Impact Factor: 5.9). 03/2015; DOI: 10.1021/acsami.5b00169 Source: PubMed
publication descriptionAligned 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 awedge-based system allowing the production of a wide range of collagen scaffolds with unidirectional pores by directionalfreezing. 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. Thesystem permitted the growth of aligned unidirectional ice crystals over a large distance (> 2.5 cm), an insulator prolonging thefreezing 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 systemwas indicated by the construction of unidirectional scaffolds from albumin, polyvinyl alcohol (a synthetic polymer), andcollagen-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.
2-Fabrication of Novel Collagenous Dual-Layer Scaffolds
publication date2014 publication descriptionZanco Journal of Pure and Applied Sciences Vol.26, No.2, 2014
publication descriptionconstructs in vitro that resemble the stratified, lattice-like structure in tissues mimicking the structure of tissues in vivo. Novel processing techniques have been developed to manufacture reproducibly dual-layer collagen scaffolds and Hybrid Synthetic/Natural Scaffolds, with excellent adhesion and high porosities for cell seeding and large surface areas for cell attachment. These scaffolds have been used to demonstrate the feasibility of regenerating several organs. These constructs comprise of the lay down of lyophilized prefabricated collagen scaffold at –20 0C -80 0C or –196 0C on a second layer of collagen suspension of 0.8% type I collagen or vice versa, subsequently the whole composite frozen at –20 0C, -80 0C or -196 0C, then lyophilized. For the preparation of these scaffolds we relied on simple techniques without the need for expensive/customized equipment, hence allowing a methodology that can be used in every laboratory.
publication date2014 publication descriptionJournal of Surgical Research, Vol. 186, Issue 2, p514–515.
publication descriptionThe ethically justifiable indications of fetal tissue engineering as a strategy for perinatal therapy can be expanded beyond life-threatening anomalies by amniotic fluid cell-based methods, in which cell procurement poses no additional risk to the mother. Amniotic mesenchymal stem cell (aMSC)-engineered bone has been shown experimentally to be a viable alternative for craniofacial repair. In this study, we sought to compare aMSC-derived bone grafts engineered from two fundamentally distinct biodegradable matrices in a leporine model of craniofacial reconstruction, as a necessary step towards preclinical optimization of this therapeutic concept.
4-Acellular tubular grafts constructed from natural materials in vascular tissue engineering: from bench to bedside.(2013)
publication dateJan 16, 2013 publication descriptionPolymeric Biomaterials: 2 Volume Set, Third Edition Published: January 16, 2013 by CRC Press Pages 389–412
publication descriptionIn the field of vascular tissue engineering, natural materials are often incorporated into vascular grafts. For a tissue engineering approach, the general strategy is to mimic the arterial architecture, thus blue-printing the native blood vessel. Various materials and methods have been used to obtain suitable vascular grafts. This chapter focuses on the development of acellular grafts and describes the route from laboratory to clinic, including fabrication, in vitro and in vivo analysis, and clinical application. Generally, xenogeneic or allogeneic grafts are prepared by decellularisation of blood vessels. In contrast, moulding and casting techniques have been developed to construct tubular grafts of natural materials from scratch using matrix components such as collagen and elastin. In vitro criteria, like cytocompatibility and biodegradability, as well as in vivo experimentation including clinical trials are also discussed.
publication dateDec 10, 2012 publication descriptionJ Tissue Eng Regen Med. 2012 Dec 10. doi: 10.1002/term.1665
publication descriptionExtensive 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.
6-EXTRACELLULAR MATRIX-BASED SCAFFOLDS FROM SCRATCH
publication dateFeb 2012 publication descriptionPan Stanford Publishing
publication descriptionIt has been recognized that tissue engineering and regenerative medicine offer an alternative technique to whole organ and tissue transplantation for diseased, failed, or malfunctioning organs. To reconstruct a new tissue by tissue engineering and regenerative medicine, triad components such as (1) cells that are harvested and dissociated from the donor tissue, (2) scaffold substrates as biomaterials in which cells are attached and cultured resulting in the implantation at the desired site of the functioning tissue, and (3) growth factors that are promoting and/or preventing cell adhesion, proliferation, migration, and differentiation by upregulating or downregulating the synthesis of protein, growth factors, and receptors are required.This book focuses on the intelligent scaffolds, associated biomaterials, and manufacturing in terms of tissue engineering and regenerative medicine, from basic principles to the most recent discoveries. The areas of focus include (1) ceramic and metal scaffold, (2) intelligent hydrogel, (3) electrospinning nanofiber, (3) novel biomaterials for scaffold, (4) novel fabrication methods for scaffold, and (5) scaffolds for target organ. The book comprises theoretical, experimental, preclinical, and clinical analysis of various properties of biomaterials, research methods and preparation techniques, and some promising applications.
7-Organ-Specific Tubular Collagen-Based Scaffolds for Tissue Engineering Applications.
publication date2011 publication descriptionTissue Engineering, part C, 17(3): 327-35(2011)
publication descriptionThe body contains a number of organs characterised 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. Standardisation of moulding, casting, freezing and lyophilising 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.
publication date2011 publication descriptionTissue Engineering and Regenerative Medicine, Vol. 8, No. 5, pp 460-470 (2011)
publication description:Ethylene oxide (EtO) gassing and β- and γ-irradiation are currently used for sterilising collagen scaffolds.
During the process, scaffolds may undergo chemical and physical alterations that may compromise their structural
integrity and functional characteristics. In this study, we compared the effects of EtO gassing, and β- and γ-irradiation at 15 and 25 kGy on type I collagen fibril-based scaffolds with and without crosslinking, and with and without
heparin. Evaluation was performed using a wide range of biophysical, biochemical, morphological and biological
parameters. EtO treatment, β-irradiation and γ-irradiation did not induce morphological changes, nor did they have
an effect on the amount of primary amine groups, or the amount of heparin covalently attached to the scaffolds. Cytocompatibility was also not affected. Irradiation, however, did result in collagen degradation products, a decrease in
collagen denaturation temperature, and an increase in proteolytic degradation, all in a dose dependent fashion. These
parameters were hardly influenced by EtO treatment. Sterilisation methods had hardly any effect on tensile strength
of crosslinked scaffolds, but -surprisingly- they increased the tensile strength of non-crosslinked scaffolds. In conclusion, a number of the collagen scaffold parameters were influenced by sterilisation, whereas others were not. Irradiation had a much larger effect than EtO. However, tensile strength and cytocompatibility, important in tissue
engineering, were not negatively influenced by any of the methods. Therefore, aspects like costs, safety and practicality of use may be taken into account in the choice of sterilisation method.
9- In Vitro Evaluation of Type I Collagen-Based Scaffolds After Applying Different Sterilization Techniques
publication dateApr 2009 publication descriptionElsevier Inc.Journal of Pediatric Urology Volume 5, Supplement 1 , Page S19, April 2009
publication descriptionPurpose
Tissue engineering provides alternative solutions e.g. developing bioactive and biodegradable constructs (scaffolds) which allow tissue regeneration. To find the ultimate sterilization method for these protein-based materials is rather challenging. The aim of this study was to sterilize type I collagen-based scaffolds using different sterilization techniques and evaluated with urothelial cell culture to assess the bioactivity and biocompatibility.
Material And Methods
Scaffolds were prepared from type I collagen and crosslinked with and without heparin. The scaffolds were disinfected with ethanol or sterilized by means of β-radiation or ethylene oxide gas. The constructs were characterized with Scanning Electron Microscopy (SEM), immunofluorescence staining (IFA) and biochemical assays. An urothelial cell line was seeded on the scaffolds and cultured for 3, 7 and 14 days. Cultured scaffolds were evaluated with cell proliferation test (WST-1), SEM and Hematoxylin & Eosin staining (H&E).
Results
Different crosslinking methods resulted in differences in the degree of crosslinking and localization of heparin. Although scaffolds were sterilized, the heparin binding growth factor VEGF could be bound to the incorporated heparin. The cell cultured scaffolds revealed a trend in compatibility. β-radiated scaffolds showed higher cell proliferation then ethylene oxide sterilized scaffolds. The heparin scaffolds showed a decrease in cell proliferation.
Conclusions
β-radiation sterilized scaffolds showed the best cell morphology and proliferation. β-radiation or ethylene oxide gas sterilization did not affect heparin binding. Considering upscale manufacturing of off-the-shelve products and the empirical data from this investigation, we conclude that β-radiation is the most suitable technique.
10- An animal model for femoral artery pseudoaneurysms.
publication descriptionJ Vasc Interv Radiol.21(7):1078-83,(2010) .
publication descriptionTo 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.
publication descriptionEur Surg 42/6: 309–313 ( 2010).
publication descriptionEsophagus tissue engineering requires optimal scaffolds that can support the implanted cells and integrate with the implanted tissue. METHODS: Four collagen scaffolds were investigated in-situ in an ovine model after 8 weeks of omental implantation to assess their suitability with regards to integration and cellular infiltration for esophagus organ tissue engineering: (a) Acellular bovine pericardial collagen (BPC), (b) Acellular cross-linked (HDMI hexamethylene diisocyanate) porcine dermal collagen (PDC), (c) Porcine porous collagen (PPC) and (d) Bovine tendon dual-layered (type-1) collagen (DLC). RESULTS: BPC demonstrated minimal integration with minimal cellular infiltration. PDC was inert to both integration and cellular infiltration. PPC enabled integration to omental tissue; however, demonstrated minimal cellular infiltration. DLC exhibited both omental integration and cellular infiltration. CONCLUSION: DLC has close to ideal properties for application in esophagus tissue engineering. BPC and PPC could have limited application in esophagus tissue engineering whereas PDC has been found to be unsuitable.
12- Construction of collagen scaffolds That Mimic the three-dimensional architecture of specific tissues
publication descriptionTissue Engineering. 13, 2387 (2007).
publication descriptionEvery 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.
publication descriptionActa Biomaterials. 6, 4666-74 ( 2010) ..
publication descriptionThere 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.
publication descriptionActa Biomaterials. 27(3):1063-71 (2011).
publication descriptionSkin substitutes are of great benefit in the treatment of patients with full-thickness wounds, but there is a need for improvement with respect to wound closure with minimal contraction, early vascularisation, and elastin formation. In this study, we designed and developed an acellular double-layered skin construct, using matrix molecules and growth factors to target specific biological processes. The epidermal layer was prepared using type I collagen, heparin and FGF7, whereas the porous dermal layer was prepared using type I collagen, solubilised elastin, dermatan sulfate, heparin, FGF2 and VEGF. The construct was biochemically and morphologically characterised and evaluated in vivo using a rat full-thickness wound model. Results were compared with the commercial skin substitute IntegraDRT and untreated wounds. The epidermal layer of the construct was about 40 µm in thickness, containing 9% heparin and 0.2 µg FGF7/mg layer, localised at the periphery. The dermal layer was 2.5 mm thick, had rounded pores, and contained 10% dermatan sulfate + heparin, and 0.7 µg FGF2 + VEGF/mg layer. The double-layered skin construct was implanted in a skin defect and on day 7, 14, 28 and 112, the (remaining) wound area was photographed, excised and (immuno)histologically evaluated. The double-layered skin construct showed more cell influx, significantly less contraction and increased blood vessel formation at early time points in comparison to IntegraDRT and/or the untreated wound. At day 14, the double-layered skin construct also had the least myofibroblasts present. At day 112, the double-layered skin construct contained more elastic fibres than IntegraDRT and the untreated wound. Structures resembling hair follicles and sebaceous glands were found in the double-layered skin construct and the untreated wound, but hardly any were found in IntegraDRT. The results provide new opportunities for the application of acellular skin constructs in the treatment of surgical wounds.
15- From molecules to matrix: construction and evaluation of molecularly defined bioscaffolds.
publication descriptionAdv. Exp. Med. Biol.; 585:279-95 (2006) .
publication descriptionIn 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.
publication descriptionTissue Engineering. Part C, Methods.: 15(3):439-9 ( 2009).
publication descriptionThe 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 μm) 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.
publication descriptionJ Vasc Surg. 52(5):1330-8, Nov. (2010).
publication descriptionDespite 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.
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.
18- Preparation of differently sized injectableI collagen microscaffolds
publication descriptionJ Tissue Eng Regen Med. 5(8):665-7, (2011).
publication descriptionCollagen 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.
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