PCL-based 3D nanofibrous structure with well-designed morphology and enhanced specific surface area for tissue engineering application.

Tissue engineering opens a new horizon for biological tissue replacement applications. Scaffolds, appropriate cells, and signaling induction are the main three determinant parameters in any tissue engineering applications. Designing a suitable scaffold which can mimic the cellular inherent and natural habitation is of great importance for cellular growth and proliferation. Just like a natural extracellular matrix (ECM), scaffolds provide the cells with an environment for performing biological functions. Accordingly, vast surface area and three-dimensional nanofibrous structures are among the pivotal characteristics of functional scaffolds in tissue engineering, and enhancement of their properties is the main purpose of the present research. In our previous study, a patterned structure composed of continuous nanofibers and microparticles was introduced. In this work, a new modification is applied for adjustment of the surface area of an electrospun/electrosprayed scaffold. For this purpose, at predetermined stages during electrospinning/electrospraying, the nitrogen gas is flushed through the mesh holes of the collector in the opposite direction of the jet movement. This method has led to the formation of very thin nanofibrous layers at nitrogen flush intervals by providing a cooling effect of the sweeping nitrogen. As a consequence, a straticulated structure has been fabricated which possesses extremely high surface/volume ratio. The porosity, water absorption, and morphological analysis were conducted on the obtained scaffold. In vitro cytocompatibility assessments as well as histological analysis demonstrated that the fabricated scaffold provides a proper substrate for cellular attachment, proliferation and infiltration. These findings can be advantageous in three-dimensional tissue engineering such as bone tissue engineering applications. Furthermore, according to the advanced microstructure and vast surface area of the fabricated samples, they can be applied in many other applications, such as membrane, filtration, etc.

Exploring Intercellular Dynamics: Ultra-Weak Biophoton Emission as a Novel Indicator of Altered Cell Functions and Disease in Oligospermia Mice.

Introduction: Biophoton emission, the spontaneous release of photons from living cells, has emerged as an attractive field of research in the study of biological systems. Scientists have recently discovered that changes in biophoton emission could serve as potential indicators of pathological conditions. This intriguing phenomenon suggests that cells might communicate and interact with each other through the exchange of these faint but significant light signals. Therefore, the present study introduces intercellular relationships with biophoton release to detect normal and abnormal cell functions to further achieve cellular interactions by focusing on cell and cell arrangement in disease conditions. Methods: Twenty male mice were assigned to control and busulfan groups. Five weeks after the injection of busulfan, the testis was removed, and then the stereological techniques and TUNEL assay were applied to estimate the histopathology of the testis tissue sections. Results: The findings revealed that the ultra-weak biophoton emission in the control group was significantly lower than in the busulfan group. The oligospermia mice model showed that it significantly changed the spatial arrangement of testicular cells and notably decreased the testis volume, length of seminiferous tubules, and the number of testicular cells. The results of the TUNEL assay showed that the percentage of apoptotic cells significantly increased in the busulfan group. Conclusion: The ultra-weak biophoton emission from testis tissue was reduced in oligospermia mice. As a result, the decline of ultra-weak biophoton can indicate a change in cell arrangement, a decrease in intercellular interaction, and eventually disease.

Fabrication of nanocomposite/nanofibrous functionally graded biomimetic scaffolds for osteochondral tissue regeneration.

One of the main challenges in treating osteochondral lesions via tissue engineering approach is providing scaffolds with unique characteristics to mimic the complexity. It has led to application of heterogeneous scaffolds as a potential candidate for engineering of osteochondral tissues, in which graded multilayered-structure should promote bone and cartilage growth. By designing three-dimensional (3D)-nanofibrous scaffolds mimicking the native extracellular matrix's nanoscale structure, cells can grow in controlled conditions and regenerate the damaged tissue. In this study, novel 3D-functionality graded nanofibrous scaffolds composed of five layers based on different compositions containing polycaprolactone(PCL)/gelatin(Gel)/nanohydroxyapatite (nHA) for osteoregeneration and chitosan(Cs)/polyvinylalcohol(PVA) for chondral regeneration are introduced. This scaffold is fabricated by electrospinning technique using spring as collector to create 3D-nanofibrous scaffolds. Fourier-transform infrared spectroscopy, X-ray diffraction, energy dispersive X-ray spectroscopy, scanning electron microscopy, mechanical compression test, porosimetry, and water uptake studies were applied to study each layer's physicochemical properties and whole functionally graded scaffold. Besides, biodegradation and biological studies were done to investigate biological performance of scaffold. Results showed that each layer has a fibrous structure with continuous nanofibers with improved pore size and porosity of novel 3D scaffold (6-13 µm and 90%) compared with two-dimensional (2D) mat (2.2 µm and 19.3%) with higher water uptake capacity (about 100 times of 2D mat). Compression modulus of electrospun scaffold was increased to 78 MPa by adding nHA. The biological studies revealed that the layer designed for osteoregeneration could improve cell proliferation rate in comparison to the layer designed for chondral regeneration. These results showed such structure possesses a promising potential for the treatment of osteochondral defects.

Improved healing of critical-size femoral defect in osteoporosis rat models using 3D elastin/polycaprolactone/nHA scaffold in combination with mesenchymal stem cells.

Osteoporosis is a common bone disease that results in elevated risk of fracture, and delayed bone healing and impaired bone regeneration are implicated by this disease. In this study, Elastin/Polycaprolactone/nHA nanofibrous scaffold in combination with mesenchymal stem cells were used to regenerate bone defects. Cytotoxicity, cytocompatibility and cellular morphology were evaluated in vitro and observations revealed that an appropriate environment for cellular attachment, growth, migration, and proliferation is provided by this scaffold. At 3 months following ovariectomy (OVX), the rats were used as animal models with an induced critical size defect in the femur to evaluate the therapeutic potential of osteogenic differentiation of bone marrow mesenchymal stem cells (BM-MSCs) seeded on 3 dimension (3D) scaffolds. In this experimental study, 24 female Wistar rats were equally divided into three groups: Control, scaffold (non-seeded BM-MSC), and scaffold + cell (seeded BM-MSC) groups. 30 days after surgery, the right femur was removed, and underwent a stereological analysis and RNA extraction in order to examine the expression of Bmp-2 and Vegf genes. The results showed a significant increase in stereological parameters and expression of Bmp-2 and Vegf in scaffold and scaffold + cell groups compared to the control rats. The present study suggests that the use of the 3D Elastin/Polycaprolactone (PCL)/Nano hydroxyapatite (nHA) scaffold in combination with MSCs may improve the fracture regeneration and accelerates bone healing at the osteotomy site in rats.

Ameliorating effects of curcumin-loaded superparamagnetic iron oxide nanoparticles (SPIONs) on the mouse testis exposed to the transient hyperthermia: A molecular and stereological study.

BACKGROUND: Testicular hyperthermia can have negative effects on male fertility. Despite reported therapeutic benefits of curcumin, several factors often limit its application such as low water solubility and instable structure. Curcumin-loaded superparamagnetic iron oxide nanoparticles (SPIONs) were designed to solve its limitation of use. In the present study, we evaluated the effect of curcumin-loaded SPIONs on transient testicular hyperthermia in mouse. MATERIALS AND METHOD: A total of 18 adult male NMRI mice were divided into three groups (n = 6): I. Controls (Cont), II. Scrotal hyperthermia (Hyp), III. Scrotal hyperthermia + curcumin-loaded iron particles (240 µL) (Hyp + Cur). After seventy days, the animals were sacrificed and used for further molecular and stereological evaluations. RESULTS: Sperm count, motility and viability significantly decreased in group hyp as compared to cont group. Furthermore, Sperm DNA fragmentation and cell apoptosis in testes increased remarkably in group hyp, compared with group cont. Stereological study showed a reduction in number of spermatogenic and Leydig cells, as well as reduced weight and volume of testes in hyp group. Degenerative appearance of testes exposed to hyperthermia was also observed. In addition, higher mRNA expression of inflammatory cytokines (IL1-α, IL6, and TNF-α) was detected in group hyp compared to cont group. However, curcumin-loaded SPIONs alleviated all of the pathologic changes in the Hyp + Cur group compared to the hyp group. CONCLUSION: Here, we used nanoparticle form of curcumin in testicular hyperthermia model and showed its ameliorating effects on testes damages caused by heat stress, which can be an appropriate method to overcome the problems that limit curcumin application in cases with increased intra testicular temperature.

Artemisia annua L. as a promising medicinal plant for powerful wound healing applications.

Artemisia annua L. has been utilized for the first time in a nanofibrous wound dressing composition. The extract of this valuable plant provides anti-inflammatory, anti-bacterial and anti-microbial properties which can be considered as a promising medicinal component in therapeutic applications. In the present work, Artemisia annua L. was picked up from Gorgan forest area of Northern Iran and its extract was prepared by methanol as the extraction solvent. In the fabrication of wound dressing, Artemisia annua L. extract was mixed with gelatin and a nanofibrous structure was formed by electrospinning technique. To have a wound dressing with acceptable stability and optimum mechanical properties, this biologically active layer was formed on a PCL nanofibrous base layer. The fabricated double-layer wound dressing was analyzed chemically, structurally, mechanically and biologically. ATR-FTIR spectra of the prepared wound dressing contain functional groups of Artemisia annua L. as peroxide groups, etc. SEM micrographs of electrospun gelatin/Artemisia annua L. confirmed the successful electrospinning process for producing Artemisia annua L.-containing nanofibers with mean diameter of 242.00 ± 67.53 nm. In vitro Artemisia annua L. release study of the fabricated wound dressings suggests a sustain release over 7 days for the crosslinked sample. In addition, evaluation of the in vitro structural stability of the prepared wound dressings confirmed the stability of the crosslinked nanofibrous structures in PBS solution environment. Biological study of the Artemisia annua L.-containing wound dressing revealed no cytotoxicity, good proliferation and attachment of the seeded fibroblasts cells and acceptable antibacterial property against Staphylococcus aureus bacteria.

Sciatic nerve injury alters the spatial arrangement of neurons and glial cells in the anterior horn of the spinal cord.

The spatial arrangement of the cell is important and considered as underlying mechanism for mathematical modeling of cell to cell interaction. The ability of cells to take on the characteristics of other cells in an organism, it is important to understand the dynamical behavior of the cells. This method implements experimental parameters of the cell-cell interaction into the mathematical simulation of cell arrangement. The purpose of this research was to explore the three-dimensional spatial distribution of anterior horn cells in the rat spinal cord to examine differences after sciatic nerve injury. Sixteen Sprague-Dawley male rats were assigned to control and axotomy groups. Twelve weeks after surgery, the anterior horn was removed for first- and second-order stereological studies. Second-order stereological techniques were applied to estimate the pair correlation and cross-correlation functions using a dipole probe superimposed onto the spinal cord sections. The findings revealed 7% and 36% reductions in the mean volume and total number of motoneurons, respectively, and a 25% increase in the neuroglial cell number in the axotomized rats compared to the control rats. In contrast, the anterior horn volume remained unchanged. The results also indicated a broader gap in the pair correlation curve for the motoneurons and neuroglial cells in the axotomized rats compared to the control rats. This finding shows a negative correlation for the distribution of motoneurons and neuroglial cells in the axotomized rats. The cross-correlation curve shows a negative correlation between the motoneurons and neuroglial cells in the axotomized rats. These findings suggest that cellular structural and functional changes after sciatic nerve injury lead to the alterations in the spatial arrangement of motoneurons and neuroglial cells, finally affecting the normal function of the central nervous system. The experimental protocol was reviewed and approved by the Animal Ethics Committee of Shahid Beheshti University of Medical Sciences (approval No. IR.SBMU.MSP.REC1395.375) on October 17, 2016.

Peripheral axotomy-induced changes of motor function and histological structure of spinal anterior horn.

The aim of this study was to evaluate changes of both peripheral motor function and histology of spinal anterior horn in adult rats after unilateral sciatectomy. Ten adult healthy rats served as control group, while in the ten rat experimental group the right sciatic nerve was severed. We followed-up nerve motor function using a sciatic function index and electromyography activity of the gastrocnemious muscle. The rats of the experimental group presented the expected gross locomotor deficit and leg muscle atrophy. At 12 weeks post sciatectomy, L4 and L5 spinal cord segments were removed from the twenty rats and were analysed by istological stereological methods. In the axotomized animals volume of the anterior horn and its content of motor neurons decreased, while the content of astrocytes increased (p < 0.05). Thus, in adult rats, beside the obvious peripheral nerve disfuction, the sciatic nerve axotomy have severe consequences on the soma of the injured motor neurons in the spinal anterior horn. All these quantitative analyses may be usefull to quantify changes occurring in adult animals after axotomy and eventual management to modify the final outcomes in peripheral nerve disorders.

Novel class of collector in electrospinning device for the fabrication of 3D nanofibrous structure for large defect load-bearing tissue engineering application.

Adequate porosity, appropriate pore size, and 3D-thick shape are crucial parameters in the design of scaffolds, as they should provide the right space for cell adhesion, spreading, migration, and growth. In this work, a novel design for fabricating a 3D nanostructured scaffold by electrospinning was taken into account. Helical spring-shaped collector was purposely designed and used for electrospinning PCL fibers. Improved morphological properties and more uniform diameter distribution of collected nanofibers on the turns of helical spring-shaped collector are confirmed by SEM analysis. SEM images elaboration showed 3D pores with average diameter of 4 and 5.5 micrometer in x-y plane and z-direction, respectively. Prepared 3D scaffold possessed 99.98% porosity which led to the increased water uptake behavior in PBS at 37°C up to 10 days, and higher degradation rate compared to 2D flat structure. Uniaxial compression test on 3D scaffolds revealed an elastic modulus of 7 MPa and a stiffness of 102 MPa, together with very low hysteresis area and residual strain. In vitro cytocompatibility test with MG-63 osteoblast-like cells using AlamarBlue™ colorimetric assay, indicated a continuous increase in cell viability for the 3D structure over the test duration. SEM observation showed enhanced cells spreading and diffusion into the underneath layers for 3D scaffold. Accelerated calcium deposition in 3D substrate was confirmed by EDX analysis. Obtained morphological, physical, and mechanical properties together with in vitro cytocompatibility results, suggest this novel technique as a proper method for the fabrication of 3D nanofibrous scaffolds for the regeneration of critical-size load bearing defects. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1535-1548, 2017.

Roll-designed 3D nanofibrous scaffold suitable for the regeneration of load bearing bone defects.

In this work, an innovative and easy method for the fabrication of 3D scaffold from 2D electrospun structures is introduced. For this aim, coral microparticles were fixed inside the nanofibrous PCL/Gelatin mat and the obtained structure was post assembled into a cylindrical design. Scaffold fabrication procedure is described in detail and morphological properties, physical and mechanical characteristics and in vitro assessments of the prepared scaffold are reported. Presences of coral microparticles in the structure led to the formation of empty spaces (3D pores) between nanofibrous layers which in turn prevent the compact accumulation of nanofibers. Post-assembly of the obtained nanofibrous coral-loaded structures makes it possible to prepare a scaffold with any desired dimension (diameter and height). Existence of coral particles within the nanofibrous mats resulted in distant placement of layers toward each other in the assembling step, which in turn create vacancy in the structure for cellular migration and fluid and nutrients exchange of the scaffold with the surrounding environment. Cell morphology within the scaffolds is investigated and cytotoxicity and cytocompatibility of the structure is evaluated using Alamar blue assay. Enhancement in mineralization of the seeded cells within the prepared coral-loaded scaffolds is demonstrated by the use of SEM-EDX. Performed compression mechanical test revealed excellent modulus and stiffness values for the cylindrical samples which are comparable to those of natural bone tissue.

Novel 3D scaffold with enhanced physical and cell response properties for bone tissue regeneration, fabricated by patterned electrospinning/electrospraying.

Developing three dimensional scaffolds mimicking the nanoscale structure of native extracellular matrix is a key parameter in tissue regeneration. In this study, we aimed to introduce a novel 3D structures composed of nanofibers (NF) and micro particles (MP) and compare their efficiency with 2D nanofibrous scaffold. The conventional nanofibrous PCL scaffolds are 2D mats fabricated by the electrospinning technique, whereas the NF/MP and patterned NF/MP PCL scaffolds are three dimensional structures fabricated by a modified electrospinning/electrospraying technique. The mentioned method was carried out by varying the electrospinning solution parameters and use of a metal mesh as the collector. Detailed fabrication process and morphological properties of the fabricated structures is discussed and porosity, pore size and PBS solution absorption value of the prepared structures are reported. Compared with the 2D structure, 3D scaffolds possessed enhanced porosity and pore size which led to the significant increase in their water uptake capacity. In vitro cell experiments were carried out on the prepared structures by the use of MG-63 osteosarcoma cell line. The fabricated 3D structures offered significantly increased cell attachment, spread and diffusion which were confirmed by SEM analysis. In vitro cytocompatibility assessed by MTT colorimetric assay indicated a continuous cell proliferation over 21 days on the innovative 3D structure, while on 2D mat cell proliferation stopped at early time points. Enhanced osteogenic differentiation of the seeded MG-63 cells on 3D scaffold was confirmed by the remarkable ALP activity together with increased and accelerated calcium deposition on this structure compared to 2D mat. Massive and well distributed bone minerals formed on patterned 3D structure were shown by EDX analysis. In comparison between NF/MP quasi-3D and Patterned NF/MP 3D scaffolds, patterned structures proceeded in all of the above properties. As such, the innovative Patterned NF/MP 3D scaffold could be considered as a proper bone graft substitute for bone tissue regeneration.