Assessment of Spirulina-PCL nanofiber for the regeneration of dermal fibroblast layers

Skin is a barrier which protects injured tissues, and thus, skin regeneration is one of many important medical issues. Tissue engineering is an attractive approach to make artificial tissue or regenerate lost tissues. While constituting artificial tissues, cells must infiltrate through scaffolds, maintaining viability and proliferation. However, a three-dimensional tissue culture involves stressful environments due to several reasons such as mass or gas transport and high cell density. Once stressed, cells produce reactive oxygen species, resulting in alleviating cellular viability and activity. Spirulina is well known to have antioxidant molecules, which have been known to modulate oxidative stress to cells. Electrospun nanofiber has widely been used as a scaffold to mimic natural extracellular matrix. In this research, we assessed Spirulina extract-imbedded nanofiber as a scaffold for an artificial skin tissue. Spirulina extract was proven to positively affect viability and proliferation of mouse fibroblasts. In addition, fibroblasts infiltrated through Spirulina extract-imbedded electrospun nanofiber without cytotoxicity.     

<Emphasis Type="Italic">Spirulina</Emphasis> extract-impregnated alginate-PCL nanofiber wound dressing for skin regeneration

In recent years, nanofibers have been developed and widely used in many products, such as cosmetics and medical supplies. They can be fabricated from various synthetic or natural polymers and attached to bioactive compounds. In previous research, polycaprolactone (PCL) nanofibers containing Spirulina extract were demonstrated to be effective on dermal wound healing in a rat model. In this study, we fabricated Spirulina extract-alginate PCL nanofibers using alginate, which has hydrophilic structures capable of holding large amounts of water, to support the backbone of the nanofibers. The morphological characteristics, hydrophilicity, water absorbance, skin adhesiveness, toxicity to human keratinocyte cells (HaCaT), and Spirulina extract emission over time were assessed. Alginate improved the efficacy of Spirulina PCL nanofibers in moisture maintenance and adhesion ability, which highly affected recovery in the rat skin wound model. In conclusion, Spirulina extract-alginate PCL nanofibers could be considered a promising candidate for wound care.     

Fabrication and evaluation of poly(epsilon-caprolactone)/silk fibroin blend nanofibrous scaffold

In this study we investigated the blend electrospinning of poly(?‐caprolactone) (PCL) and silk fibroin (SF) to improve the biodegradability and biocompatibility of PCL‐based nanofibrous scaffolds. Optimal conditions to fabricate PCL/SF (50/50) blend nanofiber were established for electrospinning using formic acid as a cosolvent and three‐dimensional (3D) PCL/SF blend nanofibrous scaffolds were prepared by a modified electrospinning process using methanol coagulation bath. The physical properties of 2D PCL/SF blend nanofiber mats and 3D highly porous blend nanofibrous scaffolds were measured and compared. To evaluate cytocompatibility of the 3D blend scaffolds as compared to 3D PCL nanofibrous scaffold, normal human dermal fibroblasts were cultured. It is concluded that biodegradability and cytocompatibility could be improved for the 3D highly porous PCL/SF (50/50) blend nanofibrous scaffold prepared by blending PCL with SF in electrospinning. In addition to the blending of PCL and SF, the 3D structure and high porosity of electrospun nanofiber assemblies may also be important factors for enhancing the performance of scaffolds. © 2011 Wiley Periodicals, Inc. Biopolymers 97: 265–275, 2012.     

Degradation of Chlorpyrifos by an alkaline phosphatase from the cyanobacterium <Emphasis Type="Italic">Spirulina platensis</Emphasis>

Spirulina is a photosynthetic, filamentous, spiral-shaped, multicellular, blue-green microalga. The two most important species are Spirulina maxima and Spirulina platensis. Spirulina is considered an excellent food, lacking toxicity and having corrective properties against viral attacks, anemia, tumor growth and malnutrition. We have observed that cultures of Spirulina platensis grow in media containing up to 80 ppm of the organophosphorous pesticide, Chlorpyrifos. It was found to be due to an alkaline phosphatase (ALP) activity that was detected in cell free extracts of Spirulina platensis. This activity was purified from the cell free extracts using ammonium sulphate precipitation and gel filtration and shown to belong to the class of EC 3.1.3.1 ALP. The purified enzyme degrades 100 ppm Chlorpyrifos to 20 ppm in 1 h transforming it into its primary metabolite 3, 5, 6-trichloro-2-pyridinol. This is the first report of degradation of Chlorpyrifos by Spirulina platensis whose enzymic mechanism has been clearly identified. These findings have immense potential for harnessing Spirulina platensis in bioremediation of polluted ecosystems.     

Enhancement of proliferation and differentiation in bone marrow hematopoietic cells by Spirulina (Arthrospira) platensis in mice

This study evaluates whether Spirulina, including its components such as phycocyanin, enhances or sustains immune functions by promoting immune competent-cell proliferation or differentiation. The effects of Spirulina of a hot-water extract (SpHW), phycocyanin (Phyc), and cell-wall component extract (SpCW) on proliferation of bone marrow cells and induction of colony-forming activity in mice were investigated. The Spirulina extracts, SpHW, Phyc, and SpCW, enhanced proliferation of bone-marrow cells and induced colony-forming activity in the spleen-cell culture supernatant. Granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-3 (IL-3) were detected in the culture supernatant of the spleen cells stimulated with the Spirulina extracts. Bone marrow-cell colony formation in soft-agar assay was also significantly induced by the blood samples and the culture supernatants of the spleen and Peyer's patch cells of the mice which ingested Spirulina extracts orally for 5 weeks in in vivo study. Ratios of neutrophils and lymphocytes in the peripheral blood and bone marrow, consequently, increased in the mice. Spirulina may have potential therapeutic benefits for improvement of weakened immune functions caused by, for example, the use of anticancer drugs.     

Wound dressing based on PVA nanofiber containing silk fibroin modified with GO/ZnO nanoparticles for superficial wound healing: In vitro and in vivo evaluations

Silk fibroin (SF), extracted from Bombyx mori, has unique physicochemical properties to achieve an efficient wound dressing. In this study, reduced graphene oxide (RGO)/ZnO NPs/silk fibroin nanocomposite was made, and an innovative nanofiber of SF/polyvinyl alcohol (PVA)/RGO/ZnO NPs was ready with the electrospinning technique and successfully characterized. The results of MIC and OD analyses were used to investigate the synthesized materials' antibacterial effects and displayed that the synthesized materials could inhibit growth against Staphylococcus aureus and Escherichia coli bacteria. However, both in vitro cytotoxicity (MTT) and scratch wound studies have shown that RGO/ZnO NPs and SF/PVA/RGO/ZnO NPs are not only non-toxic to NIH 3T3 fibroblasts, but also can cause cell viability, cell proliferation, and cell migration. Furthermore, improving the synthesized nanofiber's structural properties in the presence of RGO and ZnO NPs has been confirmed by performing tensile strength, contact angle, and biodegradation analyses. Also, in a cell attachment analysis, fibroblast cells had migrated and expanded well in the nanofibrous structures. Moreover, in vivo assay, SF/PVA/RGO/ZnO NPs nanofiber treated rats and has been shown significant healing activity and tissue regeneration compared with other treated groups. Therefore, this study suggests that SF/PVA/RGO/ZnO NPs nanofiber is a hopeful wound dressing for preventing bacteria growth and improving superficial wound repair.     

Biological designer self-assembling peptide nanofiber scaffolds significantly enhance osteoblast proliferation, differentiation and 3-D migration

A class of self-assembling peptide nanofiber scaffolds has been shown to be an excellent biological material for 3-dimension cell culture and stimulating cell migration into the scaffold, as well as for repairing tissue defects in animals. We report here the development of several peptide nanofiber scaffolds designed specifically for osteoblasts. We designed one of the pure self-assembling peptide scaffolds RADA16-I through direct coupling to short biologically active motifs. The motifs included osteogenic growth peptide ALK (ALKRQGRTLYGF) bone-cell secreted-signal peptide, osteopontin cell adhesion motif DGR (DGRGDSVAYG) and 2-unit RGD binding sequence PGR (PRGDSGYRGDS). We made the new peptide scaffolds by mixing the pure RAD16 and designer-peptide solutions, and we examined the molecular integration of the mixed nanofiber scaffolds using AFM. Compared to pure RAD16 scaffold, we found that these designer peptide scaffolds significantly promoted mouse pre-osteoblast MC3T3-E1 cell proliferation. Moreover, alkaline phosphatase (ALP) activity and osteocalcin secretion, which are early and late markers for osteoblastic differentiation, were also significantly increased. We demonstrated that the designer, self-assembling peptide scaffolds promoted the proliferation and osteogenic differentiation of MC3T3-E1. Under the identical culture medium condition, confocal images unequivocally demonstrated that the designer PRG peptide scaffold stimulated cell migration into the 3-D scaffold. Our results suggest that these designer peptide scaffolds may be very useful for promoting bone tissue regeneration.     

Preparation of electrospun polycaprolactone nanofiber mats loaded with microalgal extracts

Sustainable, ecological, and biocompatible materials are emerging for the development of novel components for tissue engineering. Microalgae being one of the unique organisms on Earth to provide various novel compounds with certain bioactivities are also a good source for the development of novel tissue scaffold materials. In this study, electrospinning technique was utilized to fabricate nanofibers from polycaprolactone loaded with microalgal extracts obtained from Haematococcus pluvialis (vegetative and carotenoid producing form) and Chlorella vulgaris. The FTIR results showed that, blending microalgae with polycaprolactone give unique bands rooted from microalgae and polycaprolactone structure. The samples were not diversified from each other, however stable bands were observed. SEM analysis revealed a uniform fiber fabrication with an average diameter of 810 ± 55 nm independent from microalgal extracts. MTT assay was done on HUVEC cell lines and results showed that nanofiber mats helped cell proliferation with extended time. Biodegradation resulted with mineral accumulation on the surface of same samples however the fiber degradation was uniform. With slow but stable biodegradation characteristics, microalgal extract loaded nanofiber mats holds great potential to be novel tissue scaffold material.     

Protective effect of <Emphasis Type="Italic">Spirulina</Emphasis> against 4-nitroquinoline-1-oxide induced toxicity

In the present study, we focused on the protective effect of Spirulina against 4-nitroquinoline-1-oxide (4NQO) induced hepato and nephrotoxicity in the experimental rats. The 4NQO administration resulted in increased levels of hepatic and renal markers [Alanine Transaminase (ALT), Aspartate Transaminase (AST), Lactate Dehydrogenase (LDH), urea, creatinine and uric acid] in the serum of experimental animals. It also increased the oxidative stress resulting in increased levels of the lipid peroxidation with a concomitant decline in the levels of non enzymic [reduced glutathione (GSH)] and enzymic antioxidants [(Superoxide dismutase (SOD), Catalase (CAT), Glutathione peroxidase (GPx), and Glutathione-S-transferase (GST)] in both liver and kidney. Oral pretreatment with aqueous extract of Spirulina prevented 4NQO induced changes in the levels of hepatic and kidney diagnostic marker enzymes in the serum of experimental rats. It counteracted the 4NQO induced lipid peroxidation and maintained the hepatic and kidney antioxidant defense system at near normal in both liver and kidney. The antioxidant responsiveness mediated by Spirulina may be anticipated to have biological significance in eliminating reactive free radicals that may otherwise affect normal cell functioning and provide a scientific rationale for the use of Spirulina.     

螺旋藻-壳寡糖生物被膜抑制剂的制备、表征及活性评价

病原菌形成的生物被膜严重威胁人类健康,显著增强了病原菌的耐药性,针对生物被膜的特效药物亟待研究。从虾、蟹壳等中提取得到的壳寡糖是一种天然碱性寡糖,具有良好的杀菌效果,但其对生物被膜的抑制作用仍有待提高。螺旋藻（Spirulina,SP）是一种表面带负电荷的微藻,其与壳寡糖形成的复合物可能发挥协同增效杀灭生物被膜深处病原菌的作用。针对提升壳寡糖的抑生物被膜作用,本研究首先通过浊度法筛选得到了杀菌效果显著的壳寡糖,并通过静电吸附作用将壳寡糖与螺旋藻结合,完成螺旋藻@壳寡糖（Spriulina@Chitooligosaccharides,SP@COS）复合物的制备。通过测定zeta电位、粒径和荧光标记等方法表征了壳寡糖和螺旋藻的结合情况,紫外-可见吸收光谱（ultraviolet-visible absorbance spectroscopy,UV-Vis）结果显示出螺旋藻对壳寡糖的包封率达90%,负载率达16%。制备的SP@COS对细菌、真菌生物被膜都有明显的增效抑制作用,且这种抑制效果主要是通过深入生物被膜内部、破坏细胞结构所实现。这些结果显示了螺旋藻-壳寡糖复合物具备作为生物被膜抑制剂的潜力,为提高壳寡糖的抑生物被膜作用、解决病原菌的危害提供了理论基础与新的思路。     

Naringin‐inlaid silk fibroin/hydroxyapatite scaffold enhances human umbilical cord‐derived mesenchymal stem cell‐based bone regeneration

ObjectivesLarge bone defects are a common, debilitating clinical condition that have substantial global health and economic burden. Bone tissue engineering technology has become one of the most promising approaches for regenerating defective bones. In this study, we fabricated a naringin‐inlaid composite silk fibroin/hydroxyapatite (NG/SF/HAp) scaffold to repair bone defects.Materials and MethodsThe salt‐leaching technology was used to fabricate the NG/SF/HAp scaffold. The cytocompatibility of the NG/SF/HAp scaffold was assessed using scanning electron microscopy, live/dead cell staining and phalloidin staining. The osteogenic and angiogenic properties were assessed in vitro and in vivo.ResultsThe porous NG/SF/HAp scaffold had a well‐designed biomimetic porous structure with osteoinductive and angiogenic activities. A gene microarray identified 854 differentially expressed genes between human umbilical cord‐derived mesenchymal stem cells (hUCMSCs) cultured on SF‐nHAp scaffolds and cells cultured on NG/SF/HAp scaffolds. The underlying osteoblastic mechanism was investigated using hUCMSCs in vitro. Naringin facilitated hUCMSC ingrowth into the SF/HAp scaffold and promoted osteogenic differentiation. The osteogenic and angiogenic capabilities of cells cultured in the NG/SF/HAp scaffold were superior to those of cells cultured in the SF/HAp scaffold.ConclusionsThe data indicate the potential of the SF/HAp composite scaffold incorporating naringin for bone regeneration.     

Chitosan nanofiber scaffold enhances hepatocyte adhesion and function

To enhance cell attachment and promote liver functions of hepatocytes cultured in bioreactors, a chitosan nanofiber scaffold was designed and prepared via electrospinning. Effects of the scaffold on hepatocyte adhesion, viability and function were then investigated. Data showed that hepatocytes on chitosan nanofiber scaffold exhibited better viability and tighter cell-substrate contact than cells on regular chitosan film. In addition, urea synthesis, albumin secretion and cytochrome P450 activity of hepatocytes on chitosan nanofiber scaffold were all 1.5 to 2 folds higher than the controls. Glycogen synthesis was also increased as compared with the controls. These results suggested the potential application of this chitosan nanofiber scaffold as a suitable substratum for hepatocyte culturing in bioreactors.     

Diet alters delayed selfing,inbreeding depression,and reproductive senescence in a freshwater snail

Reproductive success is a critical fitness attribute that is directly influenced by resource availability. Here, we investigate the effects of diet‐based resource availability on three interrelated aspects of reproductive success: a change in mating system based on mate availability, consequent inbreeding depression, and the deterioration of reproductive efficiency with age (senescence). We employed a factorial experimental design using 22 full‐sib families of the hermaphroditic freshwater snail Physa acuta to explore these interactions. Individual snails were reared in one of two mate‐availability treatments (isolated [selfing] or occasionally paired [outcrossing]) and one of two diet treatments (boiled lettuce or Spirulina, an algae that is rich in protein, vitamins, and minerals). Spirulina‐fed snails initiated reproduction at a 13% earlier age and 7% larger size than lettuce‐fed snails. Spirulina also resulted in a 30% reduction in the time delay before selfing. Compared to lettuce, a diet of Spirulina increased inbreeding depression by 52% for egg hatching rate and 64% for posthatching juvenile survival. Furthermore, Spirulina led to a 15‐fold increase in the rate of reproductive senescence compared with a diet of lettuce. These transgenerational, interactive effects of diet on inbreeding depression and reproductive senescence are discussed in the context of diet‐induced phenotypic plasticity.     

<Emphasis Type="Italic">Spirulina</Emphasis> (<Emphasis Type="Italic">Arthrospira</Emphasis>) industry in Inner Mongolia of China: current status and prospects

This paper outlines an investigation on current situation of Spirulina (Arthrospira) industry in Inner Mongolia, an internal region of China with temperate continental climate. More than 20 Spirulina plants have been established in Inner Mongolia since 2001, most of which are located at Wulan Town in the Ordos Plateau. By the end of 2009, the total annual production of Spirulina in the Ordos Plateau surpassed 700 t (dw), which account for ca. 80% of the total productivity of Inner Mongolia, and ca. 20% of China. Besides abundant solar radiation and enough freshwater favorable for Spirulina production, the three technical strategies contribute to the prosperity and success of Spirulina industry in the region: (1) reducing the cost or investment by overall advantages of rich local natural resources with low cost for Spirulina production, such as alkaline lakes, coal, electricity, and sandy land; (2) controlling the culture temperature and to avoid contamination by building plastic greenhouses on raceway ponds, (3) reducing investment by simplifying the construction of the ponds and the greenhouses. As the result, the growth period of Spirulina has been prolonged from about 120 to about 165 days, the cost of Spirulina has decreased by 25–30%, and the quality of products has been enhanced substantially. Inner Mongolia is expected to become the largest base for Spirulina production not only in China, but also in the world in the near future.     

Composite 3D printed scaffold with structured electrospun nanofibers promotes chondrocyte adhesion and infiltration

Additive manufacturing, also called 3D printing, is an effective method for preparing scaffolds with defined structure and porosity. The disadvantage of the technique is the excessive smoothness of the printed fibers, which does not support cell adhesion. In the present study, a 3D printed scaffold was combined with electrospun classic or structured nanofibers to promote cell adhesion. Structured nanofibers were used to improve the infiltration of cells into the scaffold. Electrospun layers were connected to 3D printed fibers by gluing, thus enabling the fabrication of scaffolds with unlimited thickness. The composite 3D printed/nanofibrous scaffolds were seeded with primary chondrocytes and tested in vitro for cell adhesion, proliferation and differentiation. The experiment showed excellent cell infiltration, viability, and good cell proliferation. On the other hand, partial chondrocyte dedifferentiation was shown. Other materials supporting chondrogenic differentiation will be investigated in future studies.     

Comparative analysis of nonaspanin protein sequences and expression studies in zebrafish

Nonaspanins constitute a family of proteins, also called TM9SF, characterized by a large non-cytoplasmic domain and nine putative transmembrane domains. This family is highly conserved through evolution and comprises three members in Saccharomyces cerevisiae, Dictyostelium discoideum, and Drosophila melanogaster, and four members are reported in mammals (TM9SF1–TM9SF4). Genetic studies in Dictyostelium and Drosophila have shown that TM9SF members are required for adhesion and phagocytosis in innate immune response, furthermore, human TM9SF1 plays a role in the regulation of autophagy and human TM9SF4 in tumor cannibalism. Here we report that the zebrafish genome encodes five members of this family, TM9SF1–TM9SF5, which show high level of sequence conservation with the previously reported members. Expression analysis in zebrafish showed that all members are maternally expressed and continue to be present throughout embryogenesis to adults. Gene expression could not be regulated by pathogen-associated molecular patterns such as LPS, CpG, or Poly I:C. By bioinformatic analyses of 80 TM9SF protein sequences from yeast, plants, and animals, we confirmed a very conserved protein structure. An evolutionary conserved immunoreceptor tyrosine-based inhibition motif has been detected in the cytoplasmic domain between transmembrane domain (TM) 7 and TM8 in TM9SF1, TM9SF2, TM9SF4 and TM9SF5, and at the extreme C-terminal end of TM9SF4. Finally, a conserved TRAF2 binding domain could also be predicted in the cytoplasmic regions of TM9SF2, TM9SF3, TM9SF4, and TM9SF5. This confirms the hypothesis that TM9SF proteins may play a regulatory role in a specific and ancient cellular mechanism that is involved in innate immunity.     

LIVE AND DEAD SPIRULINA SP. TO REMOVE ARSENIC (V) FROM WATER

Arsenic (As)-contaminated water is a grave health hazard and its removal from water poses a great challenge. Conventional methods are associated with many shortcomings. Biosorption of arsenic using blue-green algae is an interesting alternative to conventional methods. In this article, the results of the biosorption of As(V) as AsO₄ ^{? 3} by live and dead Spirulina sp. are reported. The sorption of arsenic could be explained satisfactorily both by the Freundlich and the Langmuir isotherms. The maximum sorption capacities of live and dead Spirulina were estimated to be 525 and 402mg/g, respectively. These values are high in comparison with those reported for other arsenic sorbents. The sorption kinetics of arsenic by both live and dead Spirulina sp. could be well modeled by Lagergrens pseudosecond order-rate equation. Infrared spectra have been employed to understand how Spirulina sp. binds with arsenate. Scanning electron micrography and fluorescent microscopic images are used to discuss the extent of uptake. Preferential uptake of Cu(II), Ni(II), Cd(II), and AsO₄ ^?3 by live Spirulina sp. was investigated and explained with the help of rate constants for sorption.     

Kraft black liquor for improving the productivity of Spirulina biomass

Summary Mass cultivation of Spirulina for commercial application suffers from poor productivity when measured against laboratory results or theoretical projections. Wider applications of algal products require that this gap be reduced. Addition of eucalyptus kraft black liquor at a maximum of 0.1% to Spirulina cultures enhanced biomass productivity by at least 40%. The factors enhancing Spirulina biomass productivity were insoluble at low pH, of low molecular mass and stable to high temperature. Single addition of kraft black liquor in outdoor continuous cultures afforded sustained enhancement in biomass productivity for at least eight weeks.     

MORPHOLOGICAL REVERSION OF SPIRULINA (ARTHROSPIRA) PLATENSIS (CYANOPHYTA): FROM LINEAR TO HELICAL1

The cyanobacterium Spirulina Turpin is characterized by its regularly coiled trichomes. Under some conditions, its helical filaments can convert to abnormal morphologies, such as irregularly curved and even linear shapes, that had been considered as a permanent degeneration that could not be reversed. However, here we found that the linear filaments of Spirulina platensis Geitler could spontaneously revert to the helical form with the same morphology as the original filaments. Further studies showed that the ultrastructural, physiological, and biochemical characteristics of linear filaments were different from those of the original filaments, whereas they were the same for the reverted and the original filaments. The SDS‐PAGE analysis revealed at least four proteins or subunits related to Spirulina morphogenesis: The 21.9‐kDa and the 20.3‐kDa proteins were highly expressed in the helical filaments, whereas the 52.0‐kDa and the 31.8‐kDa proteins were highly expressed in the linear filaments. The random amplified polymorphic DNA analysis with 96 random primers showed that the genetic background of the reverted filaments was the same as that of the original filaments but distinct from that of the linear filaments. The results indicated that linear filaments of Spirulina could revert to the original morphology under certain conditions, and their other distinctive traits were regained.