Three-layered Scaffold Research Articles

Nanofiber/hydrogel composite scaffolds based on alginate sulfate and extracellular matrix for cartilage tissue engineering applications

In this study, a three-layer composite scaffold was fabricated based on co-electrospun polycaprolactone/gelatin (PCL/GT) and polyvinyl alcohol/gelatin sacrificial nanofiber, alginate/alginate sulfate hydrogel and decellularized extracellular matrix powder. The novelty of this study is the fabrication of co-electrospun PCL/GT and polyvinyl alcohol nanofibers, which are modified by the sacrificial agent method, alginate/alginate-sulfate hydrogels, and DECM powder as growth factors with the aim of cell infiltration. Comparing the results of the microscopy images of the co-electrospun nanofibers before and after the washing process revealed that the distance between nanofibers increased from 2 ± 0.3–8 ± 0.1 µm. Furthermore, an increase in the porosity of prepared nanofibers by the washing process was observed. In addition, cell morphology studies showed that after 7 days, the cells could penetrate the underlay of nanofibers. Decellularized extracellular matrix powder in a hydrogel medium improved cell adhesion. The scaffold's porosity was tested to evaluate the ability of the cells to penetrate. The results showed that the percentage of porosity in optimized nanofiber is 94.4 ± 1.4%, which increased by 14% compared to PCL/GT with the conventional electrospinning method. Moreover, by preparing the optimal multi-layer composite, the porosity percentage was about 93.3 ± 1%. Finally, to investigate cell infiltration, cell seeding was performed on the optimal multi-layer composite for 7 and 14 days. H&E and DAPI staining analysis demonstrated that the cell penetration process through the optimal nanofiber layer was successful.

An antibacterial Multi-Layered scaffold fabricated by 3D printing and electrospinning methodologies for skin tissue regeneration

A multi-layered scaffold can mimic the hierarchical structure of the skin, accelerate the wound healing, and protect the skin against contamination and infection. In this study, a three-layered (3L) scaffold was manufactured through a combination of 3D printing and electrospinning technique. A top layer of polyurethane (PU) nanofibrous coating for the prevention of micro-organism penetration was created through electrospining. The middle layer was prepared through the 3D printing of Pluronic F127-quaternized chitosan-silver nitrate nanoparticles (F127-QCS-AgNO3), as the porous absorbent and antibacterial layer. A bottom layer of core–shell nanofibrous structure of F127-mupirocin/pectin-keratin (F127-Mup/Pec-Kr) for tissue regeneration and enable antibacterial activity was coated onto the middle layer. A range of techniques were applied to fully characterize the resultant structure. The average tensile strength and elastic modulus of the 3L scaffold were measured as 0.65 ± 0.08 MPa and 9.37 ± 2.33 MPa, respectively. The release of Ag ions, mupirocin (Mup), and the antibacterial activity of the dressings was investigated. According to the results, the highest rate of cell adhesion and viability, and angiogenic potential among the studied samples were related to the 3L scaffold, which was also found to significantly accelerate the wound healing.

Electrospun gelatin-based biomimetic scaffold with spatially aligned and three-layer architectures for vascular tissue engineering

The spatial cellular alignment and multi-layer structure are vitally important for the physiological functions of natural blood vessels. However, the two features are difficult to be constructed in one scaffold simultaneously, especially in the small-diameter vascular scaffold. Here we report a general strategy to construct a gelatin-based biomimetic three-layer vascular scaffold with spatial alignment features mimicking the natural structure of blood vessels. By using a sequential electrospinning strategy combined with folding and rolling manipulation, a three-layer vascular scaffold with inner and middle layers spatially perpendicular to each other was obtained. The special features of this scaffold could fully mimic the natural multi-layer structures of blood vessels and also possess great potential for spatial arrangement guidance of corresponding cells in blood vessels.

Development of three-layer collagen scaffolds to spatially direct tissue-specific cell differentiation for enthesis repair.

Enthesis repair remains a challenging clinical indication. Herein, a three-layer scaffold composed of a tendon-like layer of collagen type I, a fibrocartilage-like layer of collagen type II and a bone-like layer of collagen type I and hydroxyapatite, was designed to recapitulate the matrix composition of the enthesis. To aid tenogenic and fibrochondrogenic differentiation, bioactive molecules were loaded in the tendon-like layer or the fibrocartilage-like layer and their effect was assessed in in vitro setting using human bone marrow derived mesenchymal stromal cells and in an ex vivo model. Seeded human bone marrow mesenchymal stromal cells infiltrated and homogeneously spread throughout the scaffold. As a response to the composition of the scaffold, cells differentiated in a localised manner towards the osteogenic lineage and, in combination with differentiation medium, towards the fibrocartilage lineage. Whilst functionalisation of the tendon-like layer did not improve tenogenic cell commitment within the time frame of this work, relevant fibrochondrogenic markers were detected in the fibrocartilage-like layer when scaffolds were functionalised with bone morphogenetic protein 2 or non-functionalised at all, in vitro and ex vivo, respectively. Altogether, our data advocate the use of compartmentalised scaffolds for the repair and regeneration of interfacial tissues, such as enthesis.

Biomimetic three-layer hierarchical scaffolds for efficient water management and cell recruitment

Taking inspiration from the structures of roots, stems and leaves of trees in nature, a biomimetic three-layered scaffold was designed for efficient water management and cell recruitment. Using polycaprolactone (PCL) and polyacrylonitrile (PAN) as raw materials, radially oriented nanofiber films and multistage adjustable nanofiber films were prepared through electrospinning technology as the base skin-friendly layer (roots) and middle unidirectional moisture conductive material (stems), the porous polyurethane foam was integrated as the outer moisturizing layer (leaves). Among which, radially oriented nanofiber films could promote the directional migration of fibroblasts and induce cell morphological changes. For the spatially hierarchically nanofiber films, the unidirectional transport of liquid was effectively realized. While the porous polyurethane foam membrane could absorb 9 times its weight in biofluid and retain moisture for up to 10 h. As a result, the biomimetic three-layered scaffolds with different structures can promote wound epithelization and drain biofluid while avoiding wound inflammation caused by excessive biofluid, which is expected to be applied in the field of skin wounds.

A biomimetic three-layered fibrin gel/PLLA nanofibers composite as a potential scaffold for articular cartilage tissue engineering application

Developing an engineered scaffold inspired by structural features of healthy articular cartilage (AC) has attracted much attention. In this study, the design and fabrication of a three-layered fiber/hydrogel scaffold in which each layer replicates the organization of a pertinent layer of AC tissue is aimed. To this end, electrospun poly-L-lactic acid (PLLA) nanofibers are prepared and fragmented into nano/micro cylinders via aminolysis. Three-layers of the scaffold, a fibrin coated fibrous layer, a fibrin gel (FG) layer incorporating chopped fibers and a FG embedding cylindrical aligned fibrous mat perpendicular to articulating surface, respectively served as an upper, middle and bottom layers, are prepared. The layers’ physicomechanical characteristics are comprehensively evaluated. Results show that optimized electrospinning set up results in the smallest fibers diameter of 367 ± 317 nm and successful aminolysis provides amine-functionalized chopped nanofibers with a mean length of 1.46 ± 0.9 µm. Static mechanical analysis of the layers demonstrates that tensile Young’s modulus of the upper layer is 152 ± 17 MPa while compressive moduli of the middle and bottom layers are 9.8 ± 3.8 and 25.3 ± 5.2 KPa, respectively and the compressive modulus of three-layered scaffold is 13.7 ± 2.5 KPa. Assessing mechanical parameters under dynamic loading also shows that adding fibrous part in the composite scaffold layers enhances viscoelastic behavior of FG. Also, incorporation of 0.25% chopped fibers into the fibrin matrix notably enhances the equilibrium water content; however, it increases in-vitro weigh loss rate from 6% to 10.5% during a seven-day period. Cytocompatibility analysis confirms that all layers possess acceptable cytocompatibility. In a conclusion, the designed three-layered composite structure successfully mimics the physicomechanical as well as microstructural features of AC and could be suggested as a potential scaffold for this tissue regeneration.

Mimicking the Composition and Structure of the Osteochondral Tissue to Fabricate a Heterogeneous Three-Layer Scaffold for the Repair of Osteochondral Defects.

Heterogeneous three-layer scaffolds were fabricated by mimicking the biochemical composition and structure of the hyaline cartilage, calcified cartilage, and subchondral bone of the osteochondral tissue for the repair of osteochondral defects. The hyaline cartilage layer was composed of collagen I (50.0 wt %) and sodium hyaluronate (50.0 wt %). The calcified cartilage layer and subchondral bone layer were composed of collagen I, sodium hyaluronate, and nanohydroxyapatite with different proportions. N-Hydroxysuccinimide/N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride was used to mediate the crosslinking reaction of the amine groups of collagen with carboxyl groups of sodium hyaluronate. The hyaline cartilage layer and calcified cartilage layer were designed as dense structures, while the subchondral bone layer was designed as a relatively loose structure by adjusting the crosslinking degree. The scaffolds displayed a uniform and interconnected porous structure and possessed a high porosity over 85%, which were conducive to cellular adhesion and proliferation. The scaffolds could remain at 50-75% after 30 days of degradation owing to crosslinking, providing enough time for the regeneration of the osteochondral tissue. Especially, the hyaline cartilage layer and calcified cartilage layer preferred to induce the proliferation of chondrocytes, while the subchondral bone layer was more conducive to the proliferation of osteoblasts. In conclusion, the heterogeneous multilayer scaffolds could serve as implant materials for osteochondral reconstruction.

Biomimetic Elastin Fiber Patch in Rat Aorta Angioplasty.

Introduction: Vascular grafts significantly contribute to advances in vascular surgery, but none of the currently available prosthetic grafts have elastin fibers similar to native arteries. We hypothesized that a novel elastin patch could be produced after a rat decellularized thoracic aorta elastin fiber scaffold is implanted subcutaneously in rats; we tested this novel elastin patch in a rat aortic arterioplasty model. Methods: Sprague–Dawley rats (200 g) were used. Rat thoracic aortae were decellularized and sectioned at a thickness of 30 μm. A single elastin fiber scaffold was fabricated as a net (5 × 5 mm2), and then a three-layer scaffold was constructed to make a new patch. The hyaluronic acid–sodium alginate (HA/SA) hydrogel was fabricated by reacting sodium SA, HA, and CaCO3, and then the hydrogel was added to the patch to secure the elastin fibers. The patches were implanted subcutaneously in rats and harvested at day 14. The elastin patches were then implanted into the same rat’s aorta and harvested at day 14; a decellularized rat thoracic aorta (TA) patch was used as a control. Sections of the retrieved patches were stained by immunohistochemistry and immunofluorescence. Results: The elastin fibers could be secured by the hydrogel. After 14 days, the subcutaneously implanted elastin patch was incorporated into the rat tissue, and H&E staining showed that new tissue had formed around the elastin patch with almost no hydrogel left. After implantation into the rat aorta and then retrieval on day 14, H&E staining showed that there was neointima and adventitia formation in both the TA and elastin patch groups. Both patches showed a similar histological structure after implantation, and immunofluorescence showed that there were CD34- and nestin-positive cells in the neointima. In both groups, the endothelial cells expressed the arterial identity markers Ephrin-B2 and dll-4; almost one-third of the cells in the neointima were PCNA-positive with rare cleaved caspase-3-positive cells. Conclusion: We demonstrated a novel approach to making elastin fiber scaffold hydrogel patches (elastin patches) and tested them in a rat aorta arterioplasty model. This patch showed a similar healing process as the decellularized TA patch; it also showed potential applications in large animals and may be a substitute for prosthetic grafts in vascular surgery.

Cell-free osteochondral scaffolds provide a substantial clinical benefit in the treatment of osteochondral defects at a minimum follow-up of 5\u2009years

PurposeThe treatment of osteochondral lesions is challenging and no consensus has been established about the best option for restoring both cartilage and subchondral bone. Multilayer collagen-hydroxyapatite scaffolds have shown promising clinical results, but the outcome at a follow-up longer than 5 years still has to be proved. The aim was to evaluate the clinical outcome of patients with a knee isolated osteochondral lesion treated with a biomimetic three-layered scaffold at a minimum 5 years of follow-up.MethodsTwenty-nine patients (23 males and 6 females, mean age 31.5 ± 11.4 years) were evaluated retrospectively before surgery, at 1 and 2 years and at last follow-up (FU). Visual Analog Scale (VAS) for pain, International Knee Documentation Committee (IKDC) Subjective Score, Tegner-Lysholm Knee Scoring Scale and Tegner Activity Level Scale were collected. Mean FU was 7.8 ± 2.0 years (min 5.1 - max 11.3). The etiology of the defect was Osteochondritis Dissecans or osteonecrosis (17 vs 12 cases).ResultsAt 12 months FU the IKDC score improved from 51.1 ± 21.7 to 80.1 ± 17.9 (p < 0.01), Tegner Lysholm Score from 59.9 ± 17.3 to 92.5 ± 9.0 (p < 0.01), VAS from 6.1 ± 2.1 to 1.7 ± 2.3 (p < 0.01) and Tegner Activity Level Scale from 1.6 ± 0.5 to 4.9 ± 1.7 (p < 0.01). The results remained stable at 24 months, while at last FU a statistically significant decrease in IKDC, Tegner Lysholm and Tegner Activity Scale was recorded, though not clinically relevant. Patients under 35 achieved statistically better outcomes.ConclusionsThe use of a cell-free collagen-hydroxyapatite osteochondral scaffold provides substantial clinical benefits in the treatment of knee osteochondral lesions at a minimum follow-up of 5 years, especially in patients younger than 35 years.Level of evidenceLevel IV.

Characterization of Bone Marrow and Wharton's Jelly Mesenchymal Stromal Cells Response on Multilayer Braided Silk and Silk/PLCL Scaffolds for Ligament Tissue Engineering.

(1) Background: A suitable scaffold with adapted mechanical and biological properties for ligament tissue engineering is still missing. (2) Methods: Different scaffold configurations were characterized in terms of morphology and a mechanical response, and their interactions with two types of stem cells (Wharton’s jelly mesenchymal stromal cells (WJ-MSCs) and bone marrow mesenchymal stromal cells (BM-MSCs)) were assessed. The scaffold configurations consisted of multilayer braids with various number of silk layers (n = 1, 2, 3), and a novel composite scaffold made of a layer of copoly(lactic acid-co-(e-caprolactone)) (PLCL) embedded between two layers of silk. (3) Results: The insertion of a PLCL layer resulted in a higher porosity and better mechanical behavior compared with pure silk scaffold. The metabolic activities of both WJ-MSCs and BM-MSCs increased from day 1 to day 7 except for the three-layer silk scaffold (S3), probably due to its lower porosity. Collagen I (Col I), collagen III (Col III) and tenascin-c (TNC) were expressed by both MSCs on all scaffolds, and expression of Col I was higher than Col III and TNC. (4) Conclusions: the silk/PLCL composite scaffolds constituted the most suitable tested configuration to support MSCs migration, proliferation and tissue synthesis towards ligament tissue engineering.

Preparation and modeling of three\u2010layered PCL/PLGA/PCL fibrous scaffolds for prolonged drug release

The authors present the preparation procedure and a computational model of a three‐layered fibrous scaffold for prolonged drug release. The scaffold, produced by emulsion/sequential electrospinning, consists of a poly(d,l-lactic-co-glycolic acid) (PLGA) fiber layer sandwiched between two poly(ε-caprolactone) (PCL) layers. Experimental results of drug release rates from the scaffold are compared with the results of the recently introduced computational finite element (FE) models for diffusive drug release from nanofibers to the three-dimensional (3D) surrounding medium. Two different FE models are used: (1) a 3D discretized continuum and fibers represented by a simple radial one-dimensional (1D) finite elements, and (2) a 3D continuum discretized by composite smeared finite elements (CSFEs) containing the fiber smeared and surrounding domains. Both models include the effects of polymer degradation and hydrophobicity (as partitioning) of the drug at the fiber/surrounding interface. The CSFE model includes a volumetric fraction of fibers and diameter distribution, and is additionally enhanced by using correction function to improve the accuracy of the model. The computational results are validated on Rhodamine B (fluorescent drug l) and other hydrophilic drugs. Agreement with experimental results proves that numerical models can serve as efficient tools for drug release to the surrounding porous medium or biological tissue. It is demonstrated that the introduced three-layered scaffold delays the drug release process and can be used for the time-controlled release of drugs in postoperative therapy.

Three-Dimensional Printing Biologically Inspired DNA-Based Gradient Scaffolds for Cartilage Tissue Regeneration.

Cartilage damage caused by aging, repeated overloading, trauma, and diseases can result in chronic pain, inflammation, stiffness, and even disability. Unlike other types of tissues (bone, skin, muscle, etc.), cartilage tissue has an extremely weak regenerative capacity. Currently, the gold standard surgical treatment for repairing cartilage damage includes autografts and allografts. However, these procedures are limited by insufficient donor sources and the potential for immunological rejection. After years of development, engineered tissue now provides a valuable artificial replacement for tissue regeneration purposes. Three-dimensional (3D) bioprinting technologies can print customizable hierarchical structures with cells. The objective of the current work was to prepare a 3D-printed three-layer gradient scaffold with lysine-functionalized rosette nanotubes (RNTK) for improving the chondrogenic differentiation of adipose-derived mesenchymal stem cells (ADSCs). Specifically, biologically inspired RNTKs were utilized in our work because they have unique surface chemistry and biomimetic nanostructure to improve cell adhesion and growth. Different ratios of gelatin methacrylate (GelMA) and poly(ethylene glycol) diacrylate (PEGDA) were printed into a three-layer GelMA-PEGDA gradient scaffold using a stereolithography-based printer, followed by coating with RNTKs. The pores and channels (∼500 μm) were observed in the scaffold. It was found that the population of ADSCs on the GelMA-PEGDA-RNTK scaffold increased by 34% compared to the GelMA-PEGDA scaffold (control). Moreover, after 3 weeks of chondrogenic differentiation, collagen II, glycosaminoglycan, and total collagen synthesis on the GelMA-PEGDA-RNTK scaffold significantly respectively increased by 59%, 71%, and 60%, as compared to the control scaffold. Gene expression of collagen II α1, SOX 9, and aggrecan in the ADSCs growing on the GelMA-PEGDA-RNTK scaffold increased by 79%, 52%, and 47% after 3 weeks, compared to the controls, respectively. These results indicated that RNTKs are a promising type of nanotubes for promoting chondrogenic differentiation, and the present 3D-printed three-layer gradient GelMA-PEGDA-RNTK scaffold shows considerable promise for future cartilage repair and regeneration.

Design, fabrication, and optimization of a dual function three-layer scaffold for controlled release of metformin hydrochloride to alleviate fibrosis and accelerate wound healing

Abnormal wound healing caused by the over-expression of collagen and fibronectin leads to fibrosis, the major complication of all treatment modalities. A three-layer nanofiber scaffold was designed, optimized, and fabricated. This scaffold comprised two supportive polycaprolactone (PCL)-chitosan layers on the sides and a polyvinyl alcohol (PVA)-metformin hydrochloride (metformin-HCl) in the middle. The physico-chemical properties of scaffold, such as mechanical characteristics, degradation, swelling, and in-vitro drug release, were evaluated. The biological tests, including cell viability in response to metformin-HCl and Tween 80, scaffold biocompatibility, cell attachment, and antibacterial activity, were further conducted. The wound healing effect of scaffold loaded with metformin-HCl (MSc+Met) was assessed in donut-shaped silicone splints in rats. Histopathological and immunohistochemical evaluation as well as mRNA expression levels of fibrosis markers were also studied.SEM images indicated a uniform, bead-less morphology and high porosity. Surface modification of scaffold by Tween 80 improved the surface hydrophilicity and enhanced the adhesion and proliferation of fibroblasts. The scar area on day 15 in MSc+Met was significantly lower than that of other groups. Histopathological and immunohistochemical evaluation revealed that group MSc+Met was the best, having significantly lower inflammation, higher angiogenesis, the smallest scar width and depth, maximum epitheliogenesis score, and the most optimal modulation of collagen density. Local administration of metformin-HCl substantially down-regulated the expression of fibrosis-involved genes: transforming growth factor (TGF-β1), collagen type 1 (Col-I), fibronectin, collagen type 3 (Col-III), and alpha-smooth muscle actin (α-SMA). Inhibiting these genes alleviates scar formation but delays wound healing; thus, an engineered scaffold was used to prevent delay in wound healing. These results provided evidence for the first time to introduce an anti-fibrogenic slow-releasing scaffold, which acts in a dual role, both alleviating fibrosis and accelerating wound healing.

Preparation and optimization of a gelatin-based biomimetic three-layered vascular scaffold.

Electrospinning is promising approach for producing biomimetic vascular scaffolds. In particular, gelatin-based electrospun scaffolds offer excellent biocompatibility. However, gelatin-based vascular scaffolds are difficult to remove from the collector after electrospinning and cross-linking agents such as glutaraldehyde, which is commonly used to improve the strength of water-soluble electrospun gelatin, are highly toxic. Herein, we present a novel electrospinning method for preparing three-layered vascular scaffolds. First, Tween 80 was added to 2,2,2-trifluoroethanol as the polymer electrospinning solution and the gelatin electrospinning solution was mixed with polylactic acid and polycaprolactone, respectively. The three-layered vascular scaffold was fabricated by electrospinning an inner layer of gelatin, a middle layer of polyester, and another outer layer of gelatin. Finally, the electrospun scaffolds were cross-linked with microbial transglutaminase and vacuum dried. Biomechanical properties of the scaffold were determined by tensile testing. Furthermore, the structure and biocompatibility of the scaffolds were assessed by hydrophilicity tests, scanning electron microscopy, and cell seeding experiments. Our results suggest the proposed electrospinning method is suitable for preparing biomimetic vascular scaffolds for cardiovascular tissue engineering applications. Moreover, this paper provides a useful reference for the preparation and optimization of vascular scaffolds.

Nanoindentation: An advanced procedure to investigate osteochondral engineered tissues

Osteochondral scaffolds are emerging as a promising alternative for articular cartilage regeneration, although with still controversial results. In particular, the restoration of the osteochondral interface remains an open challenge. The current available investigative procedures are not optimal to quantify the properties of this region, neither to evaluate the quality of the regenerated tissue with respect to the physiological one. This study investigates an advanced procedure able to quantitatively evaluate the mechanical gradient between stiff and compliant tissues, such as in the osteochondral region where the interface between hyaline and calcified cartilage (tidemark) plays an integral role in transferring articular loads from the compliant articular surface to the stiffer underlying bone. A series of nanoindentation line scans was performed along the tidemark - starting from hyaline and expanding across calcified cartilage - on histological sections derived from sheep osteochondral tissue regenerated by a three-layered biomimetic scaffold, as well as to the adjacent healthy tissue for comparative purposes. After an accurate assessment of the indentation parameters, a sigmoid curve-fit function was applied on the reduced modulus profiles to extract gap, width and regularity of the mechanical transition. The designed procedure succeeded in quantitatively assessing the transition between compliant and stiff regions, limiting experimental issues that generally affect the reliability of the indentation mechanical data, such as apex-blunt indenter tip effect, surface roughness, and influence of the substrate. Among the evaluated parameters, the mechanical gap highlighted the main difference between native and regenerated tissues. Thanks to the information retrievable through this procedure, this load transmission area can be further investigated, providing data to tailor osteochondral engineered tissues in the future.

Electrospun triple-layered PLLA/gelatin. PRGF/PLLA scaffold induces fibroblast migration.

The function of fibroblast cells in wounded areas results in reconstruction of the extra cellular matrix and consequently resolution of granulation tissue. It is suggested that the use of platelet-rich plasma can accelerate the healing process in nonhealing or slow-healing wounds. In this study, a simple and novel method has been used to fabricate an electrospun three-layered scaffold containing plasma rich in growth factor with the aim of increasing the proliferation and migration of fibroblast cells in vitro. First, plasma rich in growth factor was derived from platelet rich plasma, and then a three-layered scaffold was fabricated using PLLA nanofibers as the outer layers and plasma rich in growth factor-containing gelatin fibers as the internal layer. The growth morphology of cells seeded on this scaffold was compared to those seeded on one layered PLLA scaffold. The study of the cell growth rate on different substrates and the migration of cells in response to the drug release of multilayered scaffold was investigated by the cell quantification assay and a modified under agarose assay. Scanning electron microscopy and fluorescence images showed that cells seeded on multilayered scaffold were completely oriented 72 hours after seeding compared to those seeded on PLLA scaffold. The cell quantification assay also indicated significant increase in proliferation rate of cells seeded on three-layered scaffold compared to those seeded on PLLA scaffold and finally, monitoring cell migration proved that cells migrate significantly toward the three-layered scaffold up to 48 to 72 hours and afterwards start to show a diminished migration rate toward this scaffold.

Major G-Quadruplex Form of HIV-1 LTR Reveals a (3 + 1) Folding Topology Containing a Stem-Loop.

Nucleic acids can form noncanonical four-stranded structures called G-quadruplexes. G-quadruplex-forming sequences are found in several genomes including human and viruses. Previous studies showed that the G-rich sequence located in the U3 promoter region of the HIV-1 long terminal repeat (LTR) folds into a set of dynamically interchangeable G-quadruplex structures. G-quadruplexes formed in the LTR could act as silencer elements to regulate viral transcription. Stabilization of LTR G-quadruplexes by G-quadruplex-specific ligands resulted in decreased viral production, suggesting the possibility of targeting viral G-quadruplex structures for antiviral purposes. Among all the G-quadruplexes formed in the LTR sequence, LTR-III was shown to be the major G-quadruplex conformation in vitro. Here we report the NMR structure of LTR-III in K+ solution, revealing the formation of a unique quadruplex–duplex hybrid consisting of a three-layer (3 + 1) G-quadruplex scaffold, a 12-nt diagonal loop containing a conserved duplex-stem, a 3-nt lateral loop, a 1-nt propeller loop, and a V-shaped loop. Our structure showed several distinct features including a quadruplex–duplex junction, representing an attractive motif for drug targeting. The structure solved in this study may be used as a promising target to selectively impair the viral cycle.

A three-layered hollow tubular scaffold as an enhancement of nerve regeneration potential

A significant clinical challenge in the surgery of peripheral nervous system injured via accidents and natural disease is development of biomimetic grafts which could potentially promote nerve repair and regeneration. Although various engineered neural tissue scaffolds have been proposed to support the neural cell functions, they have not been able to instantaneously mimic the whole characteristics of endogenous microenvironment. In this study, we proposed a three-layered tubular scaffold which could provide appropriate electrical, mechanical and biological properties for peripheral nerve engineering. While the inter layer was graphene (Gr) embedded alginate-polyvinyl alcohol (AP-Gr) fibrous scaffold with well-defined anisotropy, the outer layer was double network scaffold of polycaprolactone fumarate (PCLF) and eggshell membrane (ESM). These two layers were attached together using a polycaprolactone (PCL) fibrous membrane, a middle layer, via a simple melting process. Results showed that while the electrical conductivity of the three-layered scaffold was similar to that of AP-Gr fibrous layer, the strength of the three-layered scaffold was significantly improved compared to AP-Gr and ESM-PCLF (1.5 and 1.1 times, respectively) attributed to well attachment of the two layers. As a proof-of-concept, PC12 cell attachment, proliferation, and alignment were studied on the developed three-layered scaffold. The majority of the cells (55%) aligned (<20°) along the major axis of fibers features. Furthermore, electrical stimulation revealed positive effect on the alignment of PC12 cells and change in the cell morphology. With the ease of fabrication and mechanical robustness, the three-layered scaffold of AP-Gr and ESM-PCLF might be utilized as a versatile system for the engineering of peripheral nerve tissue.

Chitosan-Based Nanofibrous Membrane Unit with Gradient Compositional and Structural Features for Mimicking Calcified Layer in Osteochondral Matrix.

Chitosan (CH), silk fibroin (SF), and hydroxyapatite (HA) were used to prepare CH/SF/HA composites and the resulting composites were electrospun into nanofibrous membrane units with gradient compositional and structural features. The optimal membrane unit was used together with CH/HA and CH/SF composites to fabricate a type of three-layer scaffold that is intended for osteochondral repair. The bottom layer of the scaffold was built with CH/HA composites and it served as a subchondral layer, the integrated nanofibrous membrane unit functioned as the middle layer for mimicking the calcified layer and the top layer was constructed using CH/SF composites for acting as a chondral layer. The nanofibrous membrane unit was found to be permeable to some molecules with limited molecular weight and was able to prevent the seeded cells from migrating cross the unit, functioning approximately like the calcified layer in the osteochondral matrix. Layered scaffolds showed abilities to promote the growth of both chondrocytes and osteoblasts that were seeded in their chondral layer and bony layer, respectively, and they were also able to support the phenotype preservation of seeded chondrocytes and the mineralization of neotissue in the bony layer. Results suggest that this type of layered scaffolds can function as an analogue of the osteochondral matrix and it has potential in osteochondral repair.

Многослойная тканеинженерная конструкция на основе биодеградируемых и биосовместимых материалов для восстановления поврежденных желчных путей (Multilayered Tissue-engineered Construct Based on Biodegradable and Biocompatible Materials for Injured Biliary Tract Repair)

Russian Abstract: Цель: Создать и оценить биологическую и физиологическую совместимость многослойной тканеинженерной конструкции желчного протока, основу которой составляет микроволокнистый каркас из биодеградируемого и биосовместимого полимера, с двухслойной адгезией на нем клеток, длительная дренажная функция которого обеспечивается его предварительной витализацией. Материал и методы: В работе использованы методы хирургического эксперимента, конфокальной и электронной микроскопии, иммуно-ферментного анализа, измерения клеточного индекса в реальном времени, гистологии, иммуногистохимии, а также комплекс методов клеточной биологии в условиях ЦКП «Регенеративная медицина». Образцы биосовместимых каркасов получены методом электроспиннинга из волокнистого поликапролактона, сополимеров молочной и гликолиевой кислот, диацетата целлюлозы и других полимеров. Методом эмульсионного электроформования были получены биосовместимые образцы микроволокнистого поликапролактона, содержащие биологически активные соединения GFP, EGF и генотерапевтический препарат «Неоваскулген» (плазмида VEGF165). Оценка высвобождения биологически активных соединений, их биологической активности in vitro и влияние на окружающие ткани проведены в лабораторных условиях, на клеточных культурах in vitro, а также при имплантации in vivo. Использовались 4 типа клеточных культур и 2 вида лабораторных животных. Проведены морфологические исследования эксплантированных образцов методами гистологии и иммуногистохимии. Для заселения биосовместимых микроволокнистых образцов использовались методы статичного, динамического и тканевого культивирования. Работа с лабораторными животными выполнялась в Центральном виварии Первого МГМУ им. И.М. Сеченова Минздрава России. Результаты и их новизна: Впервые разработан и обоснован дизайн тканеинженерной конструкции желчного протока из модифицированных и комбинированных материалов, которая предназначена для предотвращения возникновения не только ранних, но и поздних послеоперационных осложнений при восстановительных операциях на желчных путях. Определено, что физиологическая совместимость многослойных каркасов с физико-механическими свойствами, соответствующими свойствам нативных тканей, может быть достигнута при использовании метода эмульсионного электроспиннинга. Установлено, что витализация каркаса тканеинженерной конструкции препаратом «Неоваскулген» и эпидермальным фактором роста обеспечивает пролонгированный и контролируемый выход веществ по мере резорбции каркаса для обеспечения ангиогенной функционализации трансплантата. Степень и эффективность внедрения: Результаты диссертационной работы внедрены в учебный процесс на кафедре госпитальной хирургии лечебного факультета и в практическую деятельность Отдела передовых клеточных технологий Института регенеративной медицины Первого МГМУ им. И.М. Сеченова Минздрава России. Применение: Разработанные способы получения биологически и физиологически совместимых тканеинженерных конструкций могут быть использованы в качестве элементов биоконструктора для получения моделей полых эпителиальных органов. English Abstract: Design of bioengineered organs is still complicated due to the lack of understanding of biological mechanisms that stimulate physiologically relevant conditions and induce relevant cellular media. Systematic review identified qualitative and quantitative requirements for tissue-engineered construct of the common bile duct. Tissue-specific approach was used to select materials, biologics, and cells for creation of physiologically relevant construct for injured biliary tract repair. Samples of fibrous scaffolds from PCL, PGLA, PLCL, and cellulose diacetate were created by electrospinning method and evaluated with MTT assay for cytotoxicity assessment. Mechanical properties of human bile duct sample and fibrous scaffolds were evaluated for Young's modulus and for percentage of elongation. Mechanical properties of fibrous materials were evaluated in vitro during degradation and erosion in various media. Fibrous PCL-scaffolds were formed by emulsion electrospinning with incorporation of following biomolecules: Neovasculgen gene-therapy drug (VEGF165 plasmid), EGF and GFP. The fluorescent microscopy of fibers confirmed volume modification by biomolecules. ELISA-test confirmed prolonged exit of EGF biomolecules from PCL-scaffold in vitro. Implantation of Neovasculgen-modified PCL-scaffold in rats showed vascular density increase in the implantation zone. Experimental swine model of iatrogenic bile duct injury was created and follow-up implantation of tubular fibrous PCL-scaffold in porcine bile duct showed a tissue-specific physiological biocompatibility of fibrous PCL. Fibrous three-layered scaffolds from PCL and PLCL/PLGA were obtained by electrospinning method and were tested for mechanical properties. The sample of three-layered scaffold from modified fibrous PCL and PLGA with incorporation of EGF and Neovasculgen was two-side seeded with bone marrow-derived mesenchymal stem cells and bile duct epithelial cells to obtain the tissue-engineered construct.