СРАВНИТЕЛЬНЫЙ АНАЛИЗ ТРЕХМЕРНОЙ НАНОСТРУКТУРЫ ПОРИСТЫХ БИОДЕГРАДИРУЕМЫХ МАТРИКСОВ ИЗ РЕКОМБИНАНТНОГО СПИДРОИНА И ФИБРОИНА ШЕЛКА ДЛЯ РЕГЕНЕРАТИВНОЙ МЕДИЦИНЫ

Aim. To perform a comparison of three-dimensional nanostructure of porous biocompatible scaffolds made of fibroin Bombix mori and recombinant spidroin rS1/9. Materials and methods. Three-dimensional porous scaffolds were produced by salt leaching technique. The comparison of biological characteristics of the scaffolds shows that adhesion and proliferation of mouse fibroblasts in vitro on these two types of scaffolds do not differ significantly. Comparative experiments in vivo show that regeneration of bone tissue of rats is faster with implantation of recombinant spidroin scaffolds. Three-dimensional nanostructure of scaffolds and interconnectivity of nanopores were studied with scanning probe nanotomography (SPNT) to explain higher regenerative activity of spidroin-based scaffolds. Results. Significant differences were detected in the integral density and volume of pores: the integral density of nanopores detected on 2D AFM images is 46 μm–2    and calculated volume porosity is 24% in rS1/9-based scaffolds; in fibroin-based three-dimensional structures density of nanopores and calculated volume porosity were 2.4 μm–2  and 0.5%, respectively. Three-dimensional reconstruction system of nanopores and clusters of interconnected nanopores in rS1/9-based scaffolds showed that volume fraction of pores interconnected in percolation clusters is 35.3% of the total pore volume or 8.4% of the total scaffold volume. Conclusion. Scanning probe nanotomography method allows obtaining unique information about topology of micro – and nanopore systems of artificial biostructures. High regenerative activity of rS1/9-based scaffolds can be explained by higher nanoporosity of the scaffolds.Цель. Сравнение трехмерной наноструктуры пористых биосовместимых матриксов из фиброина шелка Bombyx mori и рекомбинантного спидроина rS1/9. Материалы и методы. Трехмерные пористые матриксы были получены методом выщелачивания. При сравнении биологических свойств показано, что адгезия и пролиферация мышиных фибробластов на двух видах матриксов различались незначительно. В сравнительных экспериментах in vivo показано, что регенерация костной ткани у крыс происходит быстрее при имплантации матриксов из рекомбинантного спидроина. С целью объяснения более высокой регенеративной активности матриксов из спидроина с помощью сканирующей зондовой нанотомографии была исследована трехмерная наноструктура матриксов, а также сообщаемость нанопор внутри матриксов. Результаты. Были обнаружены существенные отличия в плотности и объеме нанопор: в матриксах из rS1/9 интегральная плотность нанопор, зафиксированная в двухмерном атомно-силовом изображении, составляла 46 мкм–2, а степень пористости – 24%; в трехмерных структурах из фиброина шелка плотность нанопор и степень пористости были 2,4 мкм–2  и 0,5% соответственно. Трехмерная реконструкция системы нанопор и образованных ими кластеров в матриксах из rS1/9 показала, что объемная доля взаимосвязанных пор в перколяционных кластерах равна 35,3% от всего объема пор, что составляет 8,4% от общего объема матрикса. Выводы. Метод сканирующей зондовой нанотомографии позволяет получить уникальную информацию о топологии систем нанопор искусственных биоконструкций. При сравнительном анализе наноструктуры искусственных матриксов из регенерированного шелка и рекомбинантного спидроина, полученных методом выщелачивания, показано, что скэффолды из спидроина имеют более высокую интегральную плотность нанопор, степень пористости и объемную долю взаимосвязанных пор

Structure of RiVax: a recombinant ricin vaccine

The X-ray crystal structure (at 2.1 Å resolution) of an immunogen under development as part of a ricin vaccine for humans is presented and structure-based analysis of the results was conducted with respect to related proteins and the known determinants for inducing or suppressing the protective immune response

Electrospun magnetic composite poly-3-hydroxybutyrate/magnetite scaffolds for biomedical applications: composition, structure, magnetic properties, and biological performance

Magnetically responsive composite polymer scaffolds have good potential for a variety of biomedical applications. In this work, electrospun composite scaffolds made of polyhydroxybutyrate (PHB) and magnetite (Fe3O4) particles (MPs) were studied before and after degradation in either PBS or a lipase solution. MPs of different sizes with high saturation magnetization were synthesized by the coprecipitation method followed by coating with citric acid (CA). Nanosized MPs were prone to magnetite-maghemite phase transformation during scaffold fabrication, as revealed by Raman spectroscopy; however, for CA-functionalized nanoparticles, the main phase was found to be magnetite, with some traces of maghemite. Submicron MPs were resistant to the magnetite-maghemite phase transformation. MPs did not significantly affect the morphology and diameter of PHB fibers. The scaffolds containing CA-coated MPs lost 0.3 or 0.2% of mass in the lipase solution and PBS, respectively, whereas scaffolds doped with unmodified MPs showed no mass changes after 1 month of incubation in either medium. In all electrospun scaffolds, no alterations of the fiber morphology were observed. Possible mechanisms of the crystalline-lamellar-structure changes in hybrid PHB/Fe3O4 scaffolds during hydrolytic and enzymatic degradation are proposed. It was revealed that particle size and particle surface functionalization affect the mechanical properties of the hybrid scaffolds. The addition of unmodified MPs increased scaffolds' ultimate strength but reduced elongation at break after the biodegradation, whereas simultaneous increases in both parameters were observed for composite scaffolds doped with CA-coated MPs. The highest saturation magnetization-higher than that published in the literature-was registered for composite PHB scaffolds doped with submicron MPs. All PHB scaffolds proved to be biocompatible, and the ones doped with nanosized MPs yielded faster proliferation of rat mesenchymal stem cells. In addition, all electrospun scaffolds were able to support angiogenesis in vivo at 30 days after implantation in Wistar rats

Novel Biodegradable Polymeric Microparticles Facilitate Scarless Wound Healing by Promoting Re-epithelialization and Inhibiting Fibrosis

Despite decades of research, the goal of achieving scarless wound healing remains elusive. One of the approaches, treatment with polymeric microcarriers, was shown to promote tissue regeneration in various in vitro models of wound healing. The in vivo effects of such an approach are attributed to transferred cells with polymeric microparticles functioning merely as inert scaffolds. We aimed to establish a bioactive biopolymer carrier that would promote would healing and inhibit scar formation in the murine model of deep skin wounds. Here we characterize two candidate types of microparticles based on fibroin/gelatin or spidroin and show that both types increase re-epithelialization rate and inhibit scar formation during skin wound healing. Interestingly, the effects of these microparticles on inflammatory gene expression and cytokine production by macrophages, fibroblasts, and keratinocytes are distinct. Both types of microparticles, as well as their soluble derivatives, fibroin and spidroin, significantly reduced the expression of profibrotic factors Fgf2 and Ctgf in mouse embryonic fibroblasts. However, only fibroin/gelatin microparticles induced transient inflammatory gene expression and cytokine production leading to an influx of inflammatory Ly6C+ myeloid cells to the injection site. The ability of microparticle carriers of equal proregenerative potential to induce inflammatory response may allow their subsequent adaptation to treatment of wounds with different bioburden and fibrotic content

Novel Photosensitizers Trigger Rapid Death of Malignant Human Cells and Rodent Tumor Transplants via Lipid Photodamage and Membrane Permeabilization

BACKGROUND: Apoptotic cascades may frequently be impaired in tumor cells; therefore, the approaches to circumvent these obstacles emerge as important therapeutic modalities. METHODOLOGY/PRINCIPAL FINDINGS: Our novel derivatives of chlorin e(6), that is, its amide (compound 2) and boronated amide (compound 5) evoked no dark toxicity and demonstrated a significantly higher photosensitizing efficacy than chlorin e(6) against transplanted aggressive tumors such as B16 melanoma and M-1 sarcoma. Compound 5 showed superior therapeutic potency. Illumination with red light of mammalian tumor cells loaded with 0.1 µM of 5 caused rapid (within the initial minutes) necrosis as determined by propidium iodide staining. The laser confocal microscopy-assisted analysis of cell death revealed the following order of events: prior to illumination, 5 accumulated in Golgi cysternae, endoplasmic reticulum and in some (but not all) lysosomes. In response to light, the reactive oxygen species burst was concomitant with the drop of mitochondrial transmembrane electric potential, the dramatic changes of mitochondrial shape and the loss of integrity of mitochondria and lysosomes. Within 3-4 min post illumination, the plasma membrane became permeable for propidium iodide. Compounds 2 and 5 were one order of magnitude more potent than chlorin e(6) in photodamage of artificial liposomes monitored in a dye release assay. The latter effect depended on the content of non-saturated lipids; in liposomes consisting of saturated lipids no photodamage was detectable. The increased therapeutic efficacy of 5 compared with 2 was attributed to a striking difference in the ability of these photosensitizers to permeate through hydrophobic membrane interior as evidenced by measurements of voltage jump-induced relaxation of transmembrane current on planar lipid bilayers. CONCLUSIONS/SIGNIFICANCE: The multimembrane photodestruction and cell necrosis induced by photoactivation of 2 and 5 are directly associated with membrane permeabilization caused by lipid photodamage

COMPARATIVE ANALYSIS OF THREE-DIMENSIONAL NANOSTRUCTURE OF POROUS BIOCOMPATIBLE SCAFFOLDS MADE OF RECOMBINANT SPIDROIN AND SILK FIBROIN FOR REGENERATIVE MEDICINE

Aim. To perform a comparison of three-dimensional nanostructure of porous biocompatible scaffolds made of fibroin Bombix mori and recombinant spidroin rS1/9. Materials and methods. Three-dimensional porous scaffolds were produced by salt leaching technique. The comparison of biological characteristics of the scaffolds shows that adhesion and proliferation of mouse fibroblasts in vitro on these two types of scaffolds do not differ significantly. Comparative experiments in vivo show that regeneration of bone tissue of rats is faster with implantation of recombinant spidroin scaffolds. Three-dimensional nanostructure of scaffolds and interconnectivity of nanopores were studied with scanning probe nanotomography (SPNT) to explain higher regenerative activity of spidroin-based scaffolds. Results. Significant differences were detected in the integral density and volume of pores: the integral density of nanopores detected on 2D AFM images is 46 μm–2    and calculated volume porosity is 24% in rS1/9-based scaffolds; in fibroin-based three-dimensional structures density of nanopores and calculated volume porosity were 2.4 μm–2  and 0.5%, respectively. Three-dimensional reconstruction system of nanopores and clusters of interconnected nanopores in rS1/9-based scaffolds showed that volume fraction of pores interconnected in percolation clusters is 35.3% of the total pore volume or 8.4% of the total scaffold volume. Conclusion. Scanning probe nanotomography method allows obtaining unique information about topology of micro – and nanopore systems of artificial biostructures. High regenerative activity of rS1/9-based scaffolds can be explained by higher nanoporosity of the scaffolds

Analysis of inflammatory processes in diffuse thickening of human aorta intima

It is generally recognized that accumulation of lipids and immune inflammatory cells is an early sign of atherosclerosis. In the present study, we investigated the relationship between the deposition of lipids, immune inflammatory cell content and expression of HLA-DR molecules, and class II major histocompatibility complex (MHC) (a marker of immune activation) in diffuse intima thickening (DIT). Lipids, including triglycerides, cholesterol esters, free cholesterol, and phospholipids were studied by chromatography and Oil Red O histochemistry, as well as by electron microscopy. Immune inflammatory cells and the expression of HLA-DR were investigated by immunohistochemistry in serial sections of the same tissue samples. It has been shown that the lipids were unevenly distributed in DIT. In the juxtaluminal sublayer, lipids were detected in the cytoplasm of intima cells and the extracellular area. In the juxtamedial musculoelastic sublayer of the intima, lipids were present predominantly along elastic fibers. The positive correlation between the presence of lipids and the expression of HLA-DR was revealed (r = 0.79; p < 0.001). A positive correlation was also found between the deposition of lipids and the number of immune inflammatory cells, although correlation was different for different sublayers of the intima. In particular, the correlation between the deposition of lipids and immune inflammatory cells in the juxtaluminal sublayer of the intima was higher (r = 0.69; p < 0.001) than in the juxtamedial musculoelastic layer (r = 0.28; p < 0.001). These data support the hypothesis that the accumulation of lipids in the intima is a key factor in the initiation of inflammatory reactions. At the preatherosclerotic stage of development of this disease, earlier pathological processes associated with lipid-dependent activation of immune cells occur mainly in the juxtaluminal portion of the intima

Widespread distribution of HLA-DR-expressing cells in macroscopically undiseased intima of the human aorta : a possible role in surveillance and maintenance of vascular homeostasis

The architectonics and cell composition of the human large arteries are not sufficiently understood. The present study is the first to undertake an analysis of the distribution and quantities of HLA-DR-expressing cells in grossly undiseased human intima using immunohistochemical and immunofluorescent analysis, complemented by the advantages of confocal microscopy. The study revealed a widespread distribution of HLA-DR-expressing cells throughout the intimal space where the cells were integrated into continuous networks via long cell processes. Numbers of HLA-DR+ cells were found to be significantly larger in the middle third of the intima than in the superficial and deep intimal portions. We speculate that a widespread distribution of HLA-DR-expressing cells in the intima of normal human aorta might play a role in the surveillance and maintenance of vascular homeostasis

Fibroblasts upregulate expression of adhesion molecules and promote lymphocyte retention in 3D fibroin/gelatin scaffolds

Bioengineered scaffolds are crucial components in artificial tissue construction. In general, these scaffolds provide inert three-dimensional (3D) surfaces supporting cell growth. However, some scaffolds can affect the phenotype of cultured cells, especially, adherent stromal cells, such as fibroblasts. Here we report on unique properties of 3D fibroin/gelatin materials, which may rapidly induce expression of adhesion molecules, such as ICAM-1 and VCAM-1, in cultured primary murine embryonic fibroblasts (MEFs). In contrast, two-dimensional (2D) fibroin/gelatin films did not show significant effects on gene expression profiles in fibroblasts as compared to 3D culture conditions. Interestingly, TNF expression was induced in MEFs cultured in 3D fibroin/gelatin scaffolds, while genetic or pharmacological TNF ablation resulted in diminished ICAM-1 and VCAM-1 expression by these cells. Using selective MAPK inhibitors, we uncovered critical contribution of JNK to 3D-induced upregulation of these adhesion molecules. Moreover, we observed ICAM-1/VCAM-1-dependent adhesion of lymphocytes to fibroblasts cultured in 3D fibroin/gelatin scaffolds, but not on 2D fibroin/gelatin films, suggesting functional reprogramming in stromal cells, when exposed to 3D environment. Finally, we observed significant infiltration of lymphocytes into 3D fibroin/gelatin, but not into collagen scaffolds in vivo upon subcapsular kidney implantation in mice. Together our data highlight the important features of fibroin/gelatin scaffolds, when they are produced as 3D sponges rather than 2D films, which should be considered when using these materials for tissue engineering