Biofunctionalized nanofibers using Arthrospira (Spirulina) biomass and biopolymer.

Electrospun nanofibers composed of polymers have been extensively researched because of their scientific and technical applications. Commercially available polyhydroxybutyrate (PHB) and polyhydroxybutyrate-co-valerate (PHB-HV) copolymers are good choices for such nanofibers. We used a highly integrated method, by adjusting the properties of the spinning solutions, where the cyanophyte Arthrospira (formally Spirulina) was the single source for nanofiber biofunctionalization. We investigated nanofibers using PHB extracted from Spirulina and the bacteria Cupriavidus necator and compared the nanofibers to those made from commercially available PHB and PHB-HV. Our study assessed nanofiber formation and their selected thermal, mechanical, and optical properties. We found that nanofibers produced from Spirulina PHB and biofunctionalized with Spirulina biomass exhibited properties which were equal to or better than nanofibers made with commercially available PHB or PHB-HV. Our methodology is highly promising for nanofiber production and biofunctionalization and can be used in many industrial and life science applications.

Three-dimensional vascularization of electrospun PCL/collagen-blend nanofibrous scaffolds in vivo.

Nanofiber scaffolds have proven their various advantages for tissue engineering and have been analyzed extensively. However, to date the three-dimensional pattern of vascularization inside nanofibrous scaffolds is unknown. This study introduces a novel method to visualize and quantify vascularization of electrospun nanofibrous PCL/collagen scaffolds in 3D in vivo. Randomly spun PCL/collagen blend and parallel aligned PCL/collagen blend/PEO scaffolds were analyzed for numbers and patterns of sprouting vessels inside the constructs using microCT scans at different time points. The image data derived from the microCT scans was converted into three-dimensional vessel trees. The aligned scaffold showed a significantly smaller number of sprouting vessels but vascularization in the center of the constructs occurred considerably earlier than in the nonwoven scaffold. Thus, for the first time the actual pattern of vascularization in nanofibrous scaffolds can be visualized three-dimensionally. These results demonstrate that the 3D pattern of vessel trees could be an essential parameter to evaluate nanofiber scaffolds for their suitability for tissue engineering as well as in vivo applications in general.

Mesenchymal stem cell adherence on poly(D, L-lactide-co-glycolide) nanofibers scaffold is integrin-beta 1 receptor dependent.

Tissue engineering is a potential approach to regenerate damaged tissue by the combination and synergism among the scaffolding material, cell source and signaling factors. In the present study, mesenchymal stem cells (MSCs) were isolated from C57BL/6 mice, cultured on poly(D, L-lactide-co-glycolide) (PLGA) scaffold produced by electrospinning technique and differentiated into chondrogenic lineage. After seeding, MSCs were responsive and became flattened with fibroblast-like morphology demonstrated by the presence of actin stress fibers. Integrin-beta1 receptor blockage reduced significantly cell adhesion with loss of actin stress fibers, demonstrating the ability of PLGA nanofiber to trigger integrin receptor-mediated cell adhesion. Present data contribute to the understanding of MSCs' behavior on these biodegradable and biocompatible scaffolds that can be used as carriers in treatments involving cell transplantation.

Preparation of nanofibers containing the microalga Spirulina (Arthrospira).

Spirulina is a microalga which offers biological functions highly favorable for tissue engineering. Highly porous scaffolds can be produced by electrospinning containing biomass of Spirulina. The goal of this contribution was therefore to establish spinning conditions allowing to produce well defined nanofibers with diameters down to about 100 nm and to produce nanofibers with various concentration of the biomass for subsequent studies in tissue engineering applications. The experimental results reveal that the blend system PEO/biomass is behaved surprisingly well in electrospinning. Very thin bead-free nanofibers with diameters of about 110 nm can be produced for different biomass contents of up to 67 wt.% of the nanofibers and for PEO concentrations in the spinning solution well below 4 wt.%. These results suggest to us the use of the biomass containing nanofibers as extracellular matrices for stem cell culture and future treatment of spinal chord injury.

Collagen matrices from sponge to nano: new perspectives for tissue engineering of skeletal muscle.

BACKGROUND: Tissue engineering of vascularised skeletal muscle is a promising method for the treatment of soft tissue defects in reconstructive surgery. In this study we explored the characteristics of novel collagen and fibrin matrices for skeletal muscle tissue engineering. We analyzed the characteristics of newly developed hybrid collagen-I-fibrin-gels and collagen nanofibers as well as collagen sponges and OPLA-scaffolds. Collagen-fibrin gels were also tested with genipin as stabilizing substitute for aprotinin. RESULTS: Whereas rapid lysis and contraction of pure collagen I- or fibrin-matrices have been great problems in the past, the latter could be overcome by combining both materials. Significant proliferation of cultivated myoblasts was detected in collagen-I-fibrin matrices and collagen nanofibers. Seeding cells on parallel orientated nanofibers resulted in strongly aligned myoblasts. In contrast, common collagen sponges and OPLA-scaffolds showed less cell proliferation and in collagen sponges an increased apoptosis rate was evident. The application of genipin caused deleterious effects on primary myoblasts. CONCLUSION: Collagen I-fibrin mixtures as well as collagen nanofibers yield good proliferation rates and myogenic differentiation of primary rat myoblasts in vitro In addition, parallel orientated nanofibers enable the generation of aligned cell layers and therefore represent the most promising step towards successful engineering of skeletal muscle tissue.

Characterization of a PLLA-collagen I blend nanofiber scaffold with respect to growth and osteogenic differentiation of human mesenchymal stem cells.

We developed a nanofiber scaffold by blending PLLA with collagen I, suitable for bone regeneration. Among several PLLA-COLI ratios tested, cell growth was better enhanced when blends with a ratio of PLLA-COLI 4:1 were used. Here, growth as well as osteoblast differentiation of hMSC was improved when compared to PLLA nanofibers alone. Therefore, blending is a suitable tool to enhance PLLA nanofibers with respect to bone tissue engineering.

Influence of nanofibers on the growth and osteogenic differentiation of stem cells: a comparison of biological collagen nanofibers and synthetic PLLA fibers.

The aim of this study was to compare biological collagen I (ColI) and synthetic poly-(L: -lactide) (PLLA) nanofibers concerning their stability and ability to promote growth and osteogenic differentiation of human mesenchymal stem cells in vitro. Matrices were seeded with human stem cells and cultivated over a period of 28 days under growth and osteoinductive conditions and analyzed during the course. During this time the PLLA nanofibers remained stable while the presence of cells resulted in an attenuation of the ColI nanofiber mesh. Although there was a tendency for better growth and osteoprotegerin production of stem cells when cultured on collagen nanofibers, there was no significant difference compared to PLLA nanofibers or controls. The gene expression of alkaline phosphate, osteocalcin and collagen I diminished in the initial phase of cultivation independent of the polymer used. In the case of PLLA fibers, this gene expression normalized during the course of cultivation, whereas the presence of collagen nanofibers resulted in an increased gene expression of osteocalcin and collagen during the course of the experiment. Taken together the PLLA fibers were easier to produce, more stable and did not compromise growth and differentiation of stem cells over the course of experiment. On the other hand, collagen nanofibers supported the differentiation process to some extent. Nevertheless, the need for fixation as well as the missing stability during cell culture requires further work.

Influence of poly(L-lactic acid) nanofibers and BMP-2-containing poly(L-lactic acid) nanofibers on growth and osteogenic differentiation of human mesenchymal stem cells.

The aim of this study was to characterize synthetic poly-(L-lactic acid) (PLLA) nanofibers concerning their ability to promote growth and osteogenic differentiation of stem cells in vitro, as well as to test their suitability as a carrier system for growth factors. Fiber matrices composed of PLLA or BMP-2-incorporated PLLA were seeded with human mesenchymal stem cells and cultivated over a period of 22 days under growth and osteoinductive conditions, and analyzed during the course of culture, with respect to gene expression of alkaline phosphatase (ALP), osteocalcin (OC), and collagen I (COL-I). Furthermore, COL-I and OC deposition, as well as cell densities and proliferation, were analyzed using fluorescence microscopy. Although the presence of nanofibers diminished the dexamethasone-induced proliferation, there were no differences in cell densities or deposition of either COL-I or OC after 22 days of culture. The gene expression of ALP, OC, and COL-I decreased in the initial phase of cell cultivation on PLLA nanofibers as compared to cover slip control, but normalized during the course of cultivation. The initial down-regulation was not observed when BMP-2 was directly incorporated into PLLA nanofibers by electrospinning, indicating that growth factors like BMP-2 might survive the spinning process in a bioactive form.

The role of mesenchymal stem cells in the pathogenesis of Co-Cr-Mo particle induced aseptic loosening: an in vitro study.

Aseptic loosening is the major factor of failed arthroplasty. Among several theories the particle disease theory is commonly accepted. Different studies examined the complex interactions between wear debris and surrounding cells, especially the monocytic and osteoblastic lineage. This study was designed to elucidate the impact of cobalt-chromium-molybdenum (Co-Cr-Mo) particles on the osteoblastic differentiation and proliferation of human mesenchymal stem cells (hMSC), with respect to the disease pattern of aseptic loosening. The hMSC were incubated in the presence of Co-Cr-Mo particles in different concentrations under growth and osteoinductive conditions. Obtained cultures were analyzed, with respect to cell density and proliferation, using CASY cell count system and Ki-67 immunostaining. Osteogenic differentiation was analyzed by fluorescence microscopy using antibodies for collagen I, alcaline phosphatase, osteocalcin and osteopontin. Additionally, scanning electron microscopy was used to analyze the localisation of Co-Cr-Mo particles in the culture system. Our findings indicate that these particles were located within the hMSC. Proliferation, as well as cell density, was diminished. The remaining cells showed increased staining of osteocalcin and osteopontin, with visible differences in deposition of these proteins, indicating a deregulation of matrix formation and differentiation respectively. Therefore, it is likely that this influence of Co-Cr-Mo particles on hMSC are involved in the disease pattern of aseptic loosening.