Recombinant Spidroin Films Attenuate Individual Markers of Glucose Induced Aging in NIH 3T3 Fibroblasts.

The effect of bioresorbable materials on aging in cultured mouse NIH 3T3 fibroblasts treated with elevated glucose concentration was investigated. The cells were grown on films produced from the silkworm fibroin and rS1/9, a recombinant analog of Nephila clavipes spidroin 1. Exposure to 50 mM glucose of the cells grown on uncoated glass support resulted in the cell growth retardation. The average areas of the cells and nuclei and the percentage of apoptotic cells increased, whereas the amount of soluble collagen decreased. In contrast, on the fibroin and spidroin films, the cell density and the percentage of 5-bromo-2'-deoxyuridine (BrdU)-positive cells were higher vs. the cells grown on the glass support. The films protected NIH 3T3 fibroblasts from the glucose-induced death. The most prominent effects on the cell density, BrdU incorporation, and apoptosis prevention were observed in the cells cultured on spidroin films. Unlike the cells grown on glass support (decrease in the soluble collagen production) or fibroin (no effect), production of soluble collagen by the cells grown on spidroin films increased after cell exposure to 50 mM glucose. Molecular analysis demonstrated that 50 mM glucose upregulated phosphorylation of the NFκB heterodimer p65 subunit in the cells grown on the glass support. The treatment of cells grown on fibroin films with 5.5 mM or 50 mM glucose had no effect on p65 phosphorylation. The same treatment decreased p65 phosphorylation in the cells on the spidroin films. These results demonstrate the anti-aging efficacy of biomaterials derived from the silk proteins and suggest that spidroin is more advantageous for tissue engineering and therapy than fibroin.

Effect of Silk Fibroin on Neuroregeneration After Traumatic Brain Injury.

Traumatic brain injury is one of the leading causes of disability among the working-age population worldwide. Despite attempts to develop neuroprotective therapeutic approaches, including pharmacological or cellular technologies, significant advances in brain regeneration have not yet been achieved. Development of silk fibroin-based biomaterials represents a new frontier in neuroregenerative therapies after brain injury. In this study, we estimated the short and long-term effects of silk fibroin scaffold transplantation on traumatic brain injury and biocompatibility of this biomaterial within rat neuro-vascular cells. Silk fibroin microparticles were injected into a brain damage area 1 day after the injury. Silk fibroin affords neuroprotection as judged by diminished brain damage and recovery of long-term neurological functions. We did not detect considerable toxicity to neuro-vascular cells cultured on fibroin/fibroin-gelatin microparticles in vitro. Cultivation of primary cell cultures of neurons and astrocytes on silk fibroin matrices demonstrated their higher viability under oxygen-glucose deprivation compared to 2D conditions on plastic plates. Thus, we conclude that scaffolds based on silk fibroin can become the basis for the creation of constructs aimed to treat brain regeneration after injury.

A CdSe/ZnS quantum dot-based platform for the delivery of aluminum phthalocyanines to bacterial cells.

Enhancement of optical properties of photosensitizers by additional light-harvesting antennas is promising for the improvement of the photodynamic therapy. However, large number of parameters determine interactions of nanoparticles and photosensitizers in complex and, thus the photodynamic efficacy of the hybrid structure. In order to achieve high efficiency of energetic coupling and photodynamic activity of such complexes it is important to know the location of the photosensitizer molecule on the nanoparticle, because it affects the spectral properties of the photosensitizer and the stability of the hybrid complex in vitro/in vivo. In this work complexes of polycationic aluminum phthalocyanines and CdSe/ZnS quantum dots were obtained. We used quantum dots which outer shell consists of polymer with carboxyl groups and provides water solubility and the negative charge of the nanoparticle. We found that phthalocyanine molecules could penetrate deeply into the polymer shell of quantum dot, leading thereby to significant changes in the spectral and photodynamic properties of phthalocyanines. We also showed that noncovalent interactions between phthalocyanine and quantum dot provide possibility for a release of the phthalocyanine from the hybrid complex and its binding to both Gram-positive and Gram-negative bacterial cells. Also, detailed characterization of the nanoparticle core and shell sizes was carried out.

Induction of ICAM-1 Expression in Mouse Embryonic Fibroblasts Cultured on Fibroin-Gelatin Scaffolds.

Culturing of allogeneic or autologous cells in three-dimensional bioresorbable scaffolds is an important step in the engineering of constructs for regenerative medicine, as well as for experimental systems to study the mechanisms of cell differentiation and cell-to-cell interaction. Artificial substrates can modulate the phenotype and functional activity of immobilized cells. Investigating these changes is important for understanding the fundamental processes underlying cellular interactions in a 3D microenvironment and for improving tissue-engineered structures. In this study, we investigated the expression of the ICAM-1 adhesion molecule in mouse embryonic fibroblasts (MEF) when cultured on gelatin-fibroin scaffolds. Increased expression of ICAM-1 in MEF was detected only under 3D culture conditions both at the mRNA and protein levels. At the same time, the MEF cultured on various substrates did not oerexpress MAdCAM-1, indicating the selective effect of 3D culture conditions on ICAM-1 expression. One possible mechanism for ICAM-1 induction in MEF is associated with the activation of AP-1, since expression of c-Fos and Junb (but not cJun and Jund) was increased in MEF in 3D. When cultured under 2D conditions, the expression level of AP-1 components did not change.

Regeneration of jejunal wall defect using an implant based on silk fibroin fibers.

Regenerative properties of fibroin implant vitalized with allogeneic bone marrow cells were assessed. The study was performed using the experimental model of rat jejunum wall damage. Three weeks after surgery, we observed recovery of all layers of the jejunum wall at the site of injury and complete degradation of the implant material.

Osteogenic differentiation of mouse bone marrow stromal cells on fibroin microcarriers.

We investigated the proliferation and osteogenic differentiation of mesenchymal stem cells cultured on fibroin microcarriers. Effective cell proliferation on the surface of the microcarriers, determined by the large surface area, and the contribution of microcarrier mineralization to the stimulation of the osteogenic differentiation of mesenchymal stem cells was revealed.

Effects of Fibroin Microcarriers on Inflammation and Regeneration of Deep Skin Wounds in Mice.

The process of tissue regeneration following damage takes place with direct participation of the immune system. The use of biomaterials as scaffolds to facilitate healing of skin wounds is a new and interesting area of regenerative medicine and biomedical research. In many ways, the regenerative potential of biological material is related to its ability to modulate the inflammatory response. At the same time, all foreign materials, once implanted into a living tissue, to varying degree cause an immune reaction. The modern approach to the development of bioengineered structures for applications in regenerative medicine should be directed toward using the properties of the inflammatory response that improve healing, but do not lead to negative chronic manifestations. In this work, we studied the effect of microcarriers comprised of either fibroin or fibroin supplemented with gelatin on the dynamics of the healing, as well as inflammation, during regeneration of deep skin wounds in mice. We found that subcutaneous administration of microcarriers to the wound area resulted in uniform contraction of the wounds in mice in our experimental model, and microcarrier particles induced the infiltration of immune cells. This was associated with increased expression of proinflammatory cytokines TNF, IL-6, IL-1ß, and chemokines CXCL1 and CXCL2, which contributed to full functional recovery of the injured area and the absence of fibrosis as compared to the control group.

Changes in morphology of actin filaments and expression of alkaline phosphatase at 3D cultivation of MG-63 osteoblast-like cells on mineralized fibroin scaffolds.

3D cultivation of MG-63 osteoblast-like cells on mineralized fibroin scaffolds leads to an increase in the expression of alkaline phosphatase, an early marker of bone formation. Increased expression is associated with the actin cytoskeleton reorganization under the influence of 3D cultivation and osteogenic calcium phosphate component of the microcarrier.

Bioengineering of Artificial Lymphoid Organs.

This review addresses the issue of bioengineering of artificial lymphoid organs.Progress in this field may help to better understand the nature of the structure-function relations that exist in immune organs. Artifical lymphoid organs may also be advantageous in the therapy or correction of immunodefficiencies, autoimmune diseases, and cancer. The structural organization, development, and function of lymphoid tissue are analyzed with a focus on the role of intercellular contacts and on the cytokine signaling pathways regulating these processes. We describe various polymeric materials, as scaffolds, for artificial tissue engineering. Finally, published studies in which artificial lymphoid organs were generated are reviewed and possible future directions in the field are discussed.

Recombinant 1F9 spidroin microgels for murine full-thickness wound repairing.

The study of the stimulating effect of the microgels (MGs) based on recombinant 1F9 spidroin on the regeneration of the deep skin wound in mice was carried out. The use of spidroin MGs was shown to increase significantly the quality of healing compared to the control. The introduction of the MG in the wound edges led to recovery of all the structural elements of the skin: the epidermis, the dermis, including vascular and nervous network, in the periphery of the wound underlying muscles, and skin appendages (sebaceous and sweat glands and hair follicles) was revealed.

New Silk Fibroin-Based Bioresorbable Microcarriers.

We fabricated bioresorbable microcarriers from water solution of Bombyx mori silk fi broin. The microcarriers are 3D structures with intricate surface and pores allowing penetration of culture medium, gas exchange, and cell adhesion. Fibroin molecules form hydrophobic structures and normally have a negative charge, which stimulates migration, but inhibits cell adhesion and makes it less effective. In order to improve adhesion efficiency and velocity, gelatin (hydrophilic biopolymer with integrin-recognizing RGD sequence) was added to the microcarrier composition. The resultant bioresorbable microcarriers support adhesion and proliferation of 3T3 murine fibroblasts.

Novel 3D-microcarriers from recombinant spidroin for regenerative medicine.

Microcarriers generated from recombinant spidroin 1F9 are suitable for use as an injection material. The microcarriers were a heterogeneous mixture of microgel particles ranging from 50 to 300 µm in size with the predominance of particles of 50-150 µm. The surface of these microparticles had a complex topography and ensured efficient cultivation of primary and immortalized fibroblasts. Intradermal injections of microgel suspensions into the area of full-thickness skin wounds did not lead to the development of acute inflammation in mice; instead, they accelerated the recovery of skin tissue and stimulated neurogenesis and angiogenesis.

[Fundamental bases for the use of silk fibroin-based bioresorbable microvehicles as an example of skin regeneration in therapeutic practice].

AIM: To assess whether silk fibroin-based microvehicles (MVs) may be used to grow fibroblasts (FBs) and keratinocytes (KCs), key cellular components in skin regeneration after injury. SUBJECTS AND METHODS: Cryogrinding was applied to derive MVs from fibroin-based and fibroin- and 30% gelatin-containing composite matrices. To examine the structure of the matrices and MVs, confocal microscopy was used to conjugate the polymer with the dye tetramethylrhodamine isothiocyanate. Microparticle size distribution was estimated by granulometric analysis. 3T3 mouse FBs and cultured primary mouse KCs expressing green fluorescent protein (GFP) were used to study whether fibroin-based MVs might be suitable for growing the cells involved in skin regeneration. KC growth was analyzed by confocal laser scanning microscopy from cellular GFP expression. The proliferation rate of FBs and KCs was estimated by a MTT assay. RESULTS: There were two derived MV types: fibroin-based and fibroin and 30% gelatin-containing composite ones. On day 1, 3T3 mouse FBs on the fibroin-based gelatin-free MVs actively proliferated and the presence of gelatin in MVs diminished the proliferation of these cells. Fibroin-based MVs were shown to be suitable for the effective in vitro growth of KCs expressing cytokeratins 5 and 14, the major markers of KCs in the basal layer. Gelatin did not give rise to accelerated KC growth. The investigation has demonstrated that is possible to regulate FB proliferation on MVs, which is of great importance in delivering the cells into the site of injury since intensive proliferation of FBs may lead to the development of fibrosis and the formation of scar tissue. Balanced FB growth is essential to the creation of optimal conditions for KC growth in composite tissue-engineering constructions. CONCLUSION: The use of fibroin-based MVs is promising for the design of novel therapeutic materials and injectable cell therapy for different diseases.

Composite Scaffolds Containing Silk Fibroin, Gelatin, and Hydroxyapatite for Bone Tissue Regeneration and 3D Cell Culturing.

Three-dimensional (3D) silk fibroin scaffolds were modified with one of the major bone tissue derivatives (nano-hydroxyapatite) and/or a collagen derivative (gelatin). Adhesion and proliferation of mouse embryonic fibroblasts (MEF) within the scaffold were increased after modification with either nano-hydroxyapatite or gelatin. However, a significant increase in MEF adhesion and proliferation was observed when both additives were introduced into the scaffold. Such modified composite scaffolds provide a new and better platform to study wound healing, bone and other tissue regeneration, as well as artificial organ bioengineering. This system can further be applied to establish experimental models to study cell-substrate interactions, cell migration and other complex processes, which may be difficult to address using the conventional two-dimensional culture systems.

[Analysis of the inflammatory processes in the diffuse thickening of the intima of human aorta].

It is generally recognized that the accumulation of lipids and immuno-inflammatory cells are early signs of atherosclerosis. In the present study, we have investigated the relationship between the deposition of lipids, of immuno-inflammatory cells and the expression of HLA-DR molecules (a marker of immune activation), the molecules of the class II of major histocompatibility complex (MHC) in diffuse thickening of the intima (DIT). Lipids, including triglycerides, cholesterol esters, free cholesterol and phospholipids were studied by chromatography, Oil Red O histochemisty, as well as by electron microscopic analysis. Immuno-inflammatory cells and the expression of HLA-DR were investigated by immunohistochemistry in consecutive section of the same tissue samples. It has been shown that the lipids were unevenly distributed in DIT. In juxtaluminal sublayer, lipids were detected both in the cytoplasm of intimal cells and extracellularly. 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). Also, a positive correlation was found between the deposition of lipids and the number of immune-inflammatory cells, although correlations 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 = 0.99; P < 0.001) than in the juxtamedial musculoelastic layer (r = 0.28; P < 0.001). These data support the hypothesis that postulates that the accumulation of lipids in the intima is a key factor in the initiation of inflammatory reactions. At the pre-atherosclerotic stage of the development of this disease, earlier pathological processes associated with lipid-dependent activation of immune cells occur mainly in the juxtaluminal portion of the intima.

Boronated derivatives of chlorin e(6) and fluoride-containing porphyrins as penetrating anions: a study using bilayer lipid membranes.

Boronated derivatives of porphyrins are studied extensively as promising compounds for boron-neutron capture therapy and photodynamic therapy. Understanding of the mechanism of their permeation across cell membranes is a key step in screening for the most efficient compounds. In the present work, we studied the ability of boronated derivatives of chlorin e(6) and porphyrins, which are mono-, di-, and tetra-anions, to permeate through planar bilayer lipid membranes (BLM). The translocation rate constants through the hydrophobic part of the lipid bilayer were estimated for monocarborane and its conjugate with chlorin e(6) by the method of electrical current relaxation. They were similar, 6.6 and 6.8 sec(-1), respectively. Conjugates of porphyrins carrying two and four carborane groups were shown to permeate efficiently through a BLM although they carry two charges and four charges, respectively. The rate of permeation of the tetraanion estimated by the BLM current had superlinear dependence on the BLM voltage. Because the resting potential of most mammalian cells is negative inside, it can be concluded that the presence of negatively-charged boronated groups in compounds should hinder the accumulation of the porphyrins in cells.

[In vitro fluorescence assay to study the folding of Kv ion channels].

A fluorescence assay to check the folding of potassium Kv channels expressed in vitro has been developed. For this aim, the fluorescently labeled channel blocker, recombinant agitoxin of yellow scorpion was employed. The level of expression of various Kv channels in vitro has been tested. It has been demonstrated that Kv2 channels form clusters on the cell surface, which are not associated with actin filaments. On the other hand, Kv10 channels form larger clusters, which are associated with actin, indicating the principal differences in the organization of cytoplasmic domains of Kv2 and Kv10 channels.

In vitro and in vivo biocompatibility studies of a recombinant analogue of spidroin 1 scaffolds.

The goal of this study was to generate porous scaffolds from the genetically engineered protein, an analogue of Nephila clavipes spidroin 1 (rS1/9) and to assess the properties of new rS1/9 scaffolds essential for bioengineering. The salt leaching technique was used to make the rS1/9 scaffolds of interconnected macroporous structure with spontaneously formed micropores. The tensile strength of scaffolds was 18 ± 5 N/cm(2). Scaffolds were relatively stable in a phosphate buffer but degraded in oxidizing environment after 11 weeks of incubation. Applicability of the recombinant spidroin 1 as a substrate for cell culture was demonstrated by successful 3T3 cells growth on the surface of rS1/9 films (270 ± 20 cells/mm(2) vs. 97 ± 8 cells/mm(2) on the glass surface, p < 0.01). The 3T3 fibroblasts readily proliferated within the rS1/9 scaffold (from initially plated 19 ± 2 cells/mm(3) to 3800 ± 304 cells/mm(3) after 2 weeks). By this time, cells were uniformly distributed between the surface and deeper layers (27% ± 8% and 33% ± 4%, respectively; p > 0.05), whereas the initial distribution was 58% ± 7% and 11% ± 8%, respectively; p < 0.05). The rS1/9 scaffolds implanted subcutaneously into Balb/c mice were well tolerated. Over a 2-month period, the scaffolds promoted an ingrowth of de novo formed vascularized connective tissue elements and nerve fibers. Thus, scaffolds made of the novel recombinant spidroin 1 analogue are potentially applicable in tissue engineering.