Evaluating oxygen tensions related to bone marrow and matrix for msc differentiation in 2d and 3d biomimetic lamellar scaffolds

The physiological O microenvironment of mesenchymal stem cells (MSCs) and osteoblasts and the dimensionality of a substrate are known to be important in regulating cell phenotype and function. By providing the physiologically normoxic environments of bone marrow (5%) and matrix (12%), we assessed their potential to maintain stemness, induce osteogenic differentiation, and enhance the material properties in the micropatterned collagen/silk fibroin scaffolds that were produced in 2D or 3D. Expression of osterix (OSX) and vascular endothelial growth factor A (VEGFA) was significantly enhanced in the 3D scaffold in all oxygen environments. At 21% O , OSX and VEGFA expressions in the 3D scaffold were respectively 13,200 and 270 times higher than those of the 2D scaffold. Markers for assessing stemness were significantly more pronounced on tissue culture polystyrene and 2D scaffold incubated at 5% O . At 21% O , we measured significant increases in ultimate tensile strength (p < 0.0001) and Young’s modulus (p = 0.003) of the 3D scaffold compared to the 2D scaffold, whilst 5% O hindered the positive effect of cell seeding on tensile strength. In conclusion, we demonstrated that the 3D culture of MSCs in collagen/silk fibroin scaffolds provided biomimetic cues for bone progenitor cells toward differentiation and enhanced the tensile mechanical properties. 2 2 2 2

Contact guidance enhances the quality of a tissue engineered corneal stroma

Corneal stroma is a very complex structure, composed of 200 lamellae of oriented collagen fibers. This highly complex nature of cornea is known to be important for its transparency and mechanical integrity. Thus, an artificial cornea design has to take into account this complex structure. In this study, behavior of human corneal keratocytes on collagen films patterned with parallel channels was investigated. Keratocytes proliferated well on films and reached confluency after 7 days in the incubation medium. Nearly all of the cells responded to the patterns and were aligned in contrast to the cells on unpatterned surfaces. Collagen type I and keratan sulfate secreted by keratocytes on patterned films appeared to be aligned in the direction of the patterns. The films showed an intermediate degradation over the course of a month. On the whole, transparency of the films increased with degradation and decreased by the presence of the cells. The decrease was, however, low and transparency level was maintained on the patterned films while on the unpatterned films a sharp decrease in transparency was followed by an improvement. This was due to the more organized distribution of cells and the oriented secretion of extracellular matrix molecules on patterned collagen films. Thus, these results suggest that application of contact guidance in cornea tissue engineering may facilitate the remodeling process, hence decrease the rehabilitation period. © 2007 Wiley Periodicals, Inc

Biodegradable nanomats produced by electrospinning : expanding multifunctionality and potential for tissue engineering

With increasing interest in nanotechnology, development of nanofibers (n-fibers) by using the technique of electrospinning is gaining new momentum. Among important potential applications of n-fiber-based structures, scaffolds for tissue-engineering represent an advancing front. Nanoscaffolds (n-scaffolds) are closer to natural extracellular matrix (ECM) and its nanoscale fibrous structure. Although the technique of electrospinning is relatively old, various improvements have been made in the last decades to explore the spinning of submicron fibers from biodegradable polymers and to develop also multifunctional drug-releasing and bioactive scaffolds. Various factors can affect the properties of resulting nanostructures that can be classified into three main categories, namely: (1) Substrate related, (2) Apparatus related, and (3) Environment related factors. Developed n-scaffolds were tested for their cytocompatibility using different cell models and were seeded with cells for to develop tissue engineering constructs. Most importantly, studies have looked at the potential of using n-scaffolds for the development of blood vessels. There is a large area ahead for further applications and development of the field. For instance, multifunctional scaffolds that can be used as controlled delivery system do have a potential and have yet to be investigated for engineering of various tissues. So far, in vivo data on n-scaffolds are scarce, but in future reports are expected to emerge. With the convergence of the fields of nanotechnology, drug release and tissue engineering, new solutions could be found for the current limitations of tissue engineering scaffolds, which may enhance their functionality upon in vivo implantation. In this paper electrospinning process, factors affecting it, used polymers, developed n-scaffolds and their characterization are reviewed with focus on application in tissue engineering

Fabrication of cell container arrays with overlaid surface topographies

This paper presents cell culture substrates in the form of microcontainer arrays with overlaid surface topographies, and a technology for their fabrication. The new fabrication technology is based on microscale thermoforming of thin polymer films whose surfaces are topographically prepatterned on a micro- or nanoscale. For microthermoforming, we apply a new process on the basis of temporary back moulding of polymer films and use the novel concept of a perforated-sheet-like mould. Thermal micro- or nanoimprinting is applied for prepatterning. The novel cell container arrays are fabricated from polylactic acid (PLA) films. The thin-walled microcontainer structures have the shape of a spherical calotte merging into a hexagonal shape at their upper circumferential edges. In the arrays, the cell containers are arranged densely packed in honeycomb fashion. The inner surfaces of the highly curved container walls are provided with various topographical micro- and nanopatterns. For a first validation of the microcontainer arrays as in vitro cell culture substrates, C2C12 mouse premyoblasts are cultured in containers with microgrooved surfaces and shown to align along the grooves in the three-dimensional film substrates. In future stem-cell-biological and tissue engineering applications, microcontainers fabricated using the proposed technology may act as geometrically defined artificial microenvironments or niches

In vitro and transdermal penetration of PHBV micro/nanoparticles

WOS: 000336904200008PubMed ID: 24510225The purpose of this study was to develop micro and nano sized drug carriers from poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and study the cell and skin penetration of these particles. PHBV micro/nanospheres were prepared by o/w emulsion method and were stained with a fluorescent dye, Nile Red. The particles were fractionated by centrifugation to produce different sized populations. Topography was studied by SEM and average particle size and its distribution were determined with particle sizer. Cell viability assay (MTT) was carried out using L929 fibroblastic cell line, and particle penetration into the cells were studied. Transdermal permeation of PHBV micro/nanospheres and tissue reaction were studied using a BALB/c mouse model. Skin response was evaluated histologically and amount of PHBV in skin was determined by gas chromatography-mass spectrometry. The average diameters of the PHBV micro/nanosphere batches were found to be 1.9 mu m, 426 and 166 nm. Polydispersity indices showed that the size distribution of micro sized particles was broader than the smaller ones. In vitro studies showed that the cells had a normal growth trend. MTT showed no signs of particle toxicity. The 426 and 166 nm sized PHBV spheres were seen to penetrate the cell membrane. The histological sections revealed no adverse effects. In view of this data nano and micro sized PHBV particles appeared to have potential to serve as topical and transdermal drug delivery carriers for use on aged or damaged skin or in cases of skin diseases such as psoriasis, and may even be used in gene transfer to cells.Government of the Russian Federation [220, 11.G34.31.0013, MD-3112.2012.4]; EC FP7 SKINTREAT project; State Planning Organization (Turkey)Turkiye Cumhuriyeti Kalkinma BakanligiThe study was supported by the Government of the Russian Federation (Decree No. 220 of 09.04.2010) (Agreement No. 11.G34.31.0013) and (Grant No MD-3112.2012.4). We gratefully acknowledge the EC FP7 SKINTREAT project and the State Planning Organization (Turkey) for the grant to establish BIOMATEN. Mr. A. Buyuksungur is acknowledged for his contributions with CLSM

Formation Of Supramolecular Structures By Negatively Charged Liposomes In The Presence Of Nucleic Acids And Divalent Cations

Cationic liposomes are being increasingly studied as delivery vehicles for bioactive agents such as DNA and other polynucleotides, The mechanism of interaction of DNA with liposomes and the organization of these interacting structures during and after the interaction are still poorly understood. Nucleic acids are known to induce aggregation and size enlargement of liposomes, In the case of phosphatidylcholine (PC) vesicles, these processes depend on the presence and concentration of divalent metal cations and the amount of cholesterol in the liposomes. In this study, anionic small unilamellar vesicles (SUV) and multilamellar vesicles (MLV) composed of dicetylphosphate (DCP):PC:cholesterol at 2:7:1 molar ratios were prepared and incubated with the DNA (from wheat) and Ca2+ (50 mM) at 25 degrees C with the aim of transferring the genetic material into the liposomes by inducing fusion of liposome-liposome aggregates created in the presence, and with the help, of DNA, The organization and the nature of the resultant liposome-DNA-Ca2+ complexes were investigated by scanning tunneling microscopy (STM) and fluorescence microscopy, Observations of complexes with similar appearances with both SUV and MLV, as shown by two quite different microscopic approaches, prove that the resultant forms are real and not artifacts of the methodology used. At this stage it is not clear whether the detected complexes represent an intermediate state before fusion of liposomes which will lead to engulfing of the genomic material by the fused liposomes, or the final form. In either case the structures consisting of some adhered or semifused liposomes bearing the nucleic acid seem to be candidates as vehicles for in-vitro and in-vivo transfection.WoSScopu