Evaluation of the Morphological Effects of Hydroxyapatite Nanoparticles on the Rheological Properties and Printability of Hydroxyapatite/Polycaprolactone Nanocomposite Inks and Final Scaffold Features.

This study is focused on the importance of nanohydroxyapatite (nHA) particle morphology with the same particle size range on the rheological behavior of polycaprolactone (PCL) composite ink with nHA as a promising candidate for additive manufacturing technologies. Two different physiologic-like nHA morphologies, that is, plate and rod shape, with particles size less than 100 nm were used. nHA powders were well characterized and the printing inks were prepared by adding the different ratios of nHA powders to 50% w/v of PCL solution (nHA/PCL: 35/65, 45/55, 55/45, and 65/35 w/w%). Subsequently, the influence of nHA particle morphology and concentration on the printability and rheological properties of composite inks was investigated. HA nanopowder analysis revealed significant differences in their microstructural properties, which affected remarkably the composite ink printability in several ways. For instance, adding up to 65% w/w of plate-like nHA to the PCL solution was possible, while nanorod HA could not be added above 45% w/w. The printed constructs were successfully fabricated using the extrusion-based printing method and had a porous structure with interconnected pores. Total porosity and surface area increased with nHA content due to the improved fiber stability following deposition of material ink. Consequently, degradation rate and bioactivity increased, while compressive properties decreased. While nanorod HA particles had a more significant impact on the mechanical strength than plate-like morphology, the latter showed less crystalline order, which makes them more bioactive than nanorod HA. It is therefore important to note that the nHA microstructure broadly affects the printability of printing ink and should be considered according to the intended biomedical applications.

miR-219 overexpressing oligodendrocyte progenitor cells for treating compression spinal cord injury.

Oligodendrocyte progenitor cells (OPCs) transplantation has been considered a promising treatment for spinal cord injury, according to previous studies. Recent research shed light on the importance of microRNA 219 (miR-219) in oligodendrocyte development, so here miR-219-overexpressing OPCs (miR-219 OPCs) were transplanted in animal models of spinal cord injury to evaluate the impact of miR-219 on oligodendrocyte differentiation and functional recovery in vivo. Our findings demonstrate that transplanted cells were distributed in the tissue sections and contributed to reducing the size of cavity in the injury site. Interestingly, miR-219 promoted OPC differentiation into mature oligodendrocyte expressing MBP in vivo whereas in absence of miR-219, less number of cells differentiated into mature oligodendrocytes. An eight week evaluation using the Basso Beattie Bresnahan (BBB) locomotor test confirmed improvement in functional recovery of hind limbs. Overall, this study demonstrated that miR-219 promoted differentiation and maturation of OPCs after transplantation and can be used in cell therapy of spinal cord injury.

Fibrin hydrogel as a scaffold for differentiation of induced pluripotent stem cells into oligodendrocytes.

The importance of tissue engineering has been established as a promising approach in treating neurodegenerative diseases. The purpose of the current study is to determine the effect of fibrin hydrogel on the differentiation of iPSC into oligodendrocyte. For this purpose, iPSCs transduced by miR-338 expressing lentiviruses. They were treated with basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and platelet-derived growth factor (PDGF)-AA. The process was traced by a 6-day treatment in a mitogen-free medium. At the end of the process, multipolar preoligodendrocytes appeared. In comparison to tissue culture plate (TCP), MTT assay demonstrated a significant increase in the viability of cells cultured in fibrin hydrogel. SEM analysis showed cells with elongated morphology and intertwined intercellular interactions. An immunofluorescent assay confirmed the expression of oligodendrocyte markers Olig2 and O4. In comparison to TCP, real-time PCR data indicated a significant increase in the expression of some markers such as Olig2, MBP, Sox10, and PDGFRα on cells encapsulated in fibrin hydrogel. Overall, the results suggest that fibrin hydrogel improves viability of cells and promotes the differentiation of iPSCs into preoligodendrocytes. Hence, it can be used as an appropriate option in the tissue engineering in order to treat neurodegenerative diseases. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 108B:192-200, 2020.

Biological evaluation of porous nanocomposite scaffolds based on strontium substituted ß-TCP and bioactive glass: An in vitro and in vivo study.

In the current study, in vitro analysis of the osteogenic potential of different scaffolds based on strontium-substituted ß-TCP (Sr-TCP) and bioactive glass (BG) ceramics was conducted using rabbit bone marrow-derived mesenchymal stem cells (rBMSCs) and the osteogenic ability of the prepared Sr-TCP and BG scaffold was evaluated through alkaline phosphatase activity, mineral deposition by Alizarin red staining, and osteoblastic gene expression experiments. The obtained in vitro results revealed that among experimental Sr-TCP/BG nanocomposite scaffold samples with the composition of Sr-TCP/BG: 100/0, 50/50, 75/25, and 25/75, the 50Sr-TCP/50BG sample presented better osteoinductive properties. Therefore, the optimized 50Sr-TCP/50BG nanocomposite scaffold was chosen for further in vivo experiments. In vivo implantation of 50Sr-TCP/50BG scaffold and hydroxyapatite (HA)/TCP granules in a rabbit calvarial defect model showed slow degradation of 50Sr-TCP/50BG scaffold and high resorption rate of HA/TCP granules at 5 months' post-surgery. However, the 50Sr-TCP/50BG scaffolds loaded by mesenchymal stem cells (MSCs) were mainly replaced with new bone even at 2 months post-implantation. Based on the obtained engineering and biological results, 50Sr-TCP/50BG nanocomposite scaffold containing MSCs could be considered as a promising alternative substitute even for load-bearing bone tissue engineering applications.

Derivation of preoligodendrocytes from human-induced pluripotent stem cells through overexpression of microRNA 338.

MicroRNAs (miRNAs) control gene expression at the posttranscriptional level and have a critical role in many biological processes such as oligodendrocyte differentiation. Recent studies have shown that microRNA 338 (miR-338) is overexpressed during the oligodendrocyte development process in the central nervous system; this finding indicates a potentially important role for miR-338 in oligodendrocyte development. To evaluate this assumption, we studied the effect of miR-338 overexpression on promoting the differentiation of oligodendrocyte progenitor cells (OPCs), derived from human-induced pluripotent stem cells (hiPSC), into preoligodendrocyte. hiPSCs were differentiated into OPCs after treating for 16 days with basic fibroblast growth factor (BFGF), epidermal growth factor (FGF), and platelet-derived growth factor (PDGF)-AA. Bipolar OPCs appeared and the expression of OPC-related markers, including Nestin, Olig2, Sox10, PDGFRα, and A2B5 was confirmed by real-time polymerase chain reaction (PCR) and immunofluorescence. Then, OPCs were transduced by miR-338 expressing lentivirus or were treated with triiodothyronine (T3) for 6 days. Data obtained from real-time PCR and immunofluorescence experiment indicated that preoligodendrocyte markers such as Sox10, O4, and MBP were expressed at higher levels in transduced cells with miR-338 in comparison with the T3 group. So, the overexpression of miR-338 in iPSC-derived OPCs can promote their differentiation into preoligodendrocyte which can be used in cell therapy of myelin-related diseases.

Overexpression of miR-219 promotes differentiation of human induced pluripotent stem cells into pre-oligodendrocyte.

Oligodendrocytes play critical roles in the central nervous system (CNS) thorough producing myelin sheaths around axons. There are a variety of approaches to produce oligodendrocytes in vitro and in vivo which are a subject of interest in many studies. A new approach to induce this differentiation is using microRNA 219 (miR-219). However, this new approach suffers from a lack of studies regarding the effect of miR-219 on differentiating human induced pluripotent stem cells (hiPSCs) to oligodendrocytes. This study aimed to assess the impact of miR-219-overexpression on hiPSCs. Initially, hiPSCs were induced with basic fibroblast growth factor (bFGF), epidermalgrowth factor (EGF) and platelet-derived growth factor (PDGF)-AA, then, miR-219- green fluorescent protein (GFP)-expressing lentiviruses were utilized for cell infection. Microscopic observation revealed significant morphological changes and data obtained from quantitative reverse transcription PCR and immunofluorescence analysis of differentiated cells showed that the expression of various oligodendrocyte stage-specific markers such as Nestin, Olig2, Sox10, PDGFRα, A2B5, O4, and MBP increased. In addition, higher expressions of pre-oligodendrocyte markers were detected in the cells transduced with miR-219 lentivirus in comparison with the cells treated with triiodothyronine (T3). These results suggest that overexpression of miR-219 promotes differentiation of hiPSCs to pre-oligodendrocyte cells, providing a potential source for cell therapy by replacing and restoring the lost cell function in neurodegenerative and demyelinating diseases.

Fabrication and characterization of highly porous barium titanate based scaffold coated by Gel/HA nanocomposite with high piezoelectric coefficient for bone tissue engineering applications.

It is well established that the piezoelectric effect plays an important physiological role in bone growth, remodeling and fracture healing. Barium titanate, as a well-known piezoelectric ceramic, is especially an attractive material as a scaffold for bone tissue engineering applications. In this regard, we tried to fabricate a highly porous barium titanate based scaffolds by foam replication method and polarize them by applying an external electric field. In order to enhance the mechanical and biological properties, polarized/non-polarized scaffolds were coated with gelatin and nanostructured HA and characterized for their morphologies, porosities, piezoelectric and mechanical properties. The results showed that the compressive strength and piezoelectric coefficient of porous scaffolds increased with the increase of sintering temperature. After being coated with Gel/HA nanocomposite, the interconnected porous structure and pore size of the scaffolds almost remain unchanged while the Gel/nHA-coated scaffolds exhibited enhanced compressive strength and elastic modulus compared with the uncoated samples. Also, the effect of polarizing and coating of optimal scaffolds on adhesion, viability, and proliferation of the MG63 osteoblast-like cell line was evaluated by scanning electron microscope (SEM) and MTT assay. The cell culture experiments revealed that developed scaffolds had good biocompatibility and cells were able to adhere, proliferate and migrate into pores of the scaffolds. Furthermore, cell density was significantly higher in the coated scaffolds at all tested time-points. These results indicated that highly porous barium titanate scaffolds coated with Gel/HA nanocomposite has great potential in tissue engineering applications for bone tissue repair and regeneration.

Repair of rat critical size calvarial defect using osteoblast-like and umbilical vein endothelial cells seeded in gelatin/hydroxyapatite scaffolds.

The present study used a previously developed three-dimensional Gelatin/Hydroxyapatite (Gel/HA) homogeneous nanocomposite scaffold with porosity of 82% and interconnecting pores ranging from 300 to 500 µm. Cell-seeded scaffolds were used to evaluate bone regeneration of rat critical-size calvarial defect. Totally, 36 male Wistar rats were randomly divided into four experimental groups, including blank defect (defects without any graft), blank scaffold (defects filled with Gel/HA scaffold without cells), and two groups of cell-seeded scaffolds (defects filled with either Gel/HA scaffold seeded with osteoblast-like and endothelial cells or osteoblast-like cell-seeded constructs). After 1, 4, and 12 weeks of scaffold implantation, rats were sacrificed and the calvaria were harvested for histological, immunohistochemical and histomorphometric analysis. In vitro tests showed that scaffolds were nontoxic to cells and promoted ideal cellular attachment. In vivo investigation on scaffold revealed that blank calvarial defects indicated incomplete tissue coverage and little evidence of bone healing. However, blank scaffold and cell-seeded scaffolds significantly promoted osteoconduction and ostegogenesis. Taken together, pre-seeded Gel/HA nanocomposite scaffold with osteoblasts and endothelial cells presented an effective combination to improve osteogenesis in the engineered bone implant. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1770-1778, 2016.