Efficient three-dimensional (3D) human bone differentiation on quercetin-functionalized isotropic nano-architecture chitinous patterns of cockroach wings.

Developing cost-effective, biocompatible scaffolds with nano-structured surface that truthfully replicate the physico-(bio)chemical and structural properties of bone tissue's extracellular matrix (ECM) is still challenging. In this regard, surface functionalization of natural scaffolds to enhance capability of mimicking 3D niches of the bone tissue has been suggested as a solution. In the current study, we aimed to investigate the potential of chitin-based cockroach wings (CW) as a natural scaffold for bone tissue engineering. To raise the osteogenic differentiation capacity of such a scaffold, a quercetin coating was also applied (hereafter this scaffold is referred as QCW). Moreover, the QCW scaffold exhibited effective antibacterial properties against gram-positive S. aureus bacteria. With respect to bone regeneration, the QCW scaffold optimally induced the differentiation of adipose-derived human mesenchymal stem cells (AD-hMSCs) into osteoblasts, as validated by mineralization assays, alkaline phosphatase (ALP) activity measurements, expression of pre-osteocyte marker genes, and immunocytochemical staining. Confirmation of the potent biocompatibility and physicochemical characteristics of the QCW scaffold through a series of in vitro and in vivo analysis revealed that surface modification had significant effect on multi-purpose features of obtained scaffold. Altogether, surface modification of QCW made it as an affordable bioinspired scaffold for bone tissue engineering.

Exploration of curcumin-incorporated dual anionic alginate-quince seed gum films for transdermal drug delivery.

The idea of combining bioextracted polymers for wound healing applications has emerged in hopes of developing highly flexible and mechanically stable hydrogel films with controlled drug delivery, biocompatibility, and high collagen deposition. In the present research, polysaccharide films composed of Alginate and Quince Seed Gum (QSG) were fabricated by ionic crosslinking, and their potential for curcumin delivery and wound healing were examined. In this regard, microstructure, mechanical properties, thermal stability, physiochemical properties, and biocompatibility of films with three different QSG amounts (25 %, 50 %, and 75 %) were studied. Because of the optimum properties of 25 % QSG films like better transparency (Opacity = 6.1 %), higher flexibility (Elongation = 28.9 %), less water solubility (Water solubility = 66.6 %), proper absorbance (Swelling degree = >600 %), and suitable biocompatibility (Cell viability = >85 %), they were used for drug delivery examination. Curcumin administration through films with and without stearic acid modification was investigated. Stearic Acid (SA) modified samples demonstrated superior compatibility between hydrophobic drug and hydrophilic film. Stearic acid-modified film could prolong the curcumin release up to 48 h and showed increased collagen synthesis and TGF-ß expression, making it an excellent candidate for transdermal drug delivery and wound healing applications.

Enhanced osteogenesis on proantocyanidin-loaded date palm endocarp cellulosic matrices: A novel sustainable approach for guided bone regeneration.

Developing inexpensive, biocompatible natural scaffolds that can support the differentiation and proliferation of stem cells has been recently emphasized by the research community to faster obtain the FDA approvals for regenerative medicine. In this regard, plant-derived cellulose materials are a novel class of sustainable scaffolding materials with high potentials for bone tissue engineering (BTE). However, low bioactivity of the plant-derived cellulose scaffolds restricts cell proliferation and cell differentiation. This limitation can be addressed though surface-functionalization of cellulose scaffolds with natural antioxidant polyphenols, e.g., grape seed proanthocyanidin (PCA)-rich extract (GSPE). Despite the various merits of GSPE as a natural antioxidant, its impact on the proliferation and adhesion of osteoblast precursor cells, and on their osteogenic differentiation is an as-yet unknown issue. Here, we investigated the effects of GSPE surface functionalization on the physicochemical properties of decellularized date (Phoenix dactyliferous) fruit inner layer (endocarp) (DE) scaffold. In this regard, various physiochemical characteristics of the DE-GSPE scaffold such as hydrophilicity, surface roughness, mechanical stiffness, porosity, and swelling, and biodegradation behavior were compared with those of the DE scaffold. Additionally, the impact of the GSPE treatment of the DE scaffold on the osteogenic response of human mesenchymal stem cells (hMSCs) was thoroughly studied. For this purpose, cellular activities including cell adhesion, calcium deposition and mineralization, alkaline phosphatase (ALP) activity, and expression levels of bone-related genes were monitored. Taken together, the GSPE treatment enhanced the physicochemical and biological properties of the DE-GSPE scaffold, thereby raising its potentials as a promising candidate for guided bone regeneration.

Efficient CRISPR/Cas9-Mediated BAX Gene Ablation in CHO Cells To Impair Apoptosis and Enhance Recombinant Protein Production.

Background: Despite recent advances in recombinant biotherapeutics production using CHO cells, their productivity remains lower than industrial needs, mainly due to apoptosis. Objectives: Present study aimed to exploit CRISPR/Cas9 technology to specifically disrupt the BAX gene to attenuate apoptosis in recombinant Chinese hamster's ovary cells producing erythropoietin. Materials and Methods: The STRING database was used to identify the key pro-apoptotic genes to be modified by CRISPR/Cas9 technique. The single guide RNAs (sgRNAs) targeting identified gene (BAX) were designed, and CHO cells were then transfected with vectors. Afterward, changes in the expression of the Bax gene and consequent production rates of erythropoietin were investigated in manipulated cells, even in the presence of an apoptosis inducer agent, oleuropein. Results: BAX disruption significantly prolonged cell viability and increased proliferation rate in manipulated clones (152%, P-value = 0.0002). This strategy reduced the levels of Bax protein expression in manipulated cells by more than 4.3-fold (P-value <0.0001). The Bax-8 manipulated cells displayed higher threshold tolerance to the stress and consequence apoptosis compared to the control group. Also, they exhibited a higher IC50 compared to the control in the presence of oleuropein (5095 µM.ml-1 Vs. 2505 µM.ml-1). We found a significant increase in recombinant protein production levels in manipulated cells, even in the presence of 1,000 µM oleuropein compared to the control cell line (p-value=0.0002). Conclusions: CRISPR/Cas9 assisted BAX gene ablation is promising to improve erythropoietin production in CHO cells via engineering anti-apoptotic genes. Therefore, exploiting genome editing tools such as CRISPR/Cas9 has been proposed to develop host cells that result in a safe, feasible, and robust manufacturing operation with a yield that meets the industrial requirements.

In vitro modeling of hepatocellular carcinoma niche on decellularized tomato thorny leaves: a novel natural three-dimensional (3D) scaffold for liver cancer therapeutics.

Liver cancer is now one of the main causes leading to death worldwide. To achieve reliable therapeutic effects, it is crucial to develop efficient approaches to test novel anticancer drugs. Considering the significant contribution of tumor microenvironment to cell's response to medications, in vitro 3D bioinspiration of cancer cell niches can be regarded as an advanced strategy to improve the accuracy and reliability of the drug-based treatment. In this regard, decellularized plant tissues can perform as suitable 3D scaffolds for mammalian cell culture to create a near-to-real condition to test drug efficacy. Here, we developed a novel 3D natural scaffold made from decellularized tomato hairy leaves (hereafter called as DTL) to mimic the microenvironment of human hepatocellular carcinoma (HCC) for pharmaceutical purposes. The surface hydrophilicity, mechanical properties, and topography measurement and molecular analyses revealed that the 3D DTL scaffold is an ideal candidate for liver cancer modeling. The cells exhibited a higher growth and proliferation rate within the DTL scaffold, as verified by quantifying the expression of related genes, DAPI staining, and SEM imaging of the cells. Moreover, prilocaine, an anticancer drug, showed a higher effectiveness against the cancer cells cultured on the 3D DTL scaffold, compared to a 2D platform. Taken together, this new cellulosic 3D scaffold can be confidently proposed for chemotherapeutic testing of drugs on hepatocellular carcinoma.

Crossing Phylums: Butterfly Wing as a Natural Perfusable Three-Dimensional (3D) Bioconstruct for Bone Tissue Engineering.

Despite the advent of promising technologies in tissue engineering, finding a biomimetic 3D bio-construct capable of enhancing cell attachment, maintenance, and function is still a challenge in producing tailorable scaffolds for bone regeneration. Here, osteostimulatory effects of the butterfly wings as a naturally porous and non-toxic chitinous scaffold on mesenchymal stromal cells are assessed. The topographical characterization of the butterfly wings implied their ability to mimic bone tissue microenvironment, whereas their regenerative potential was validated after a 14-day cell culture. In vivo analysis showed that the scaffold induced no major inflammatory response in Wistar rats. Topographical features of the bioconstruct upregulated the osteogenic genes, including COL1A1, ALP, BGLAP, SPP1, SP7, and AML3 in differentiated cells compared to the cells cultured in the culture plate. However, butterfly wings were shown to provide a biomimetic microstructure and proper bone regenerative capacity through a unique combination of various structural and material properties. Therefore, this novel platform can be confidently recommended for bone tissue engineering applications.

Computational and in vitro validation of cardiogenic induction of quercetin on adipose-derived mesenchymal stromal cells through the inhibition of Wnt and non-Smad-dependent TGF-ß pathways.

Exploiting human mesenchymal stem cells (hMSCs) was proposed as a promising therapeutic approach for cardiovascular disease due to their capacity to differentiate into cardiac cells. Though modulation of the intracellular signaling pathways dominantly WNT/ß catenin and transforming growth factor-ß (TGF-ß) have been reported to promote differentiation of hMSCs into cardiomyocytes in the prevailing literature, a safe and reproducible system for their clinical application has not yet turned into reality. In the present study, the molecular docking-based strategy was first applied for evaluating the potency of some natural phenolic compounds in the modulation of Wnt and TGF-ß signaling pathways using a vital class of crystallographic protein structures of WNT signaling regulators such as Frizzled, Disheveled, GSK3-ß, ß-catenin, LRP 5/6 extracellular domain, Tankyrase and their variety of active pockets. Then, the impacts of plant-derived chemical compounds on the regulation of the relevant signals for the differentiation of hMSCs into the definitive mesoderm lineage and cardiac progenitors were assessed in vitro. Data obtained revealed the synergistic activity of Wnt and TGF-ß superfamily to direct cardiac differentiation in human cardiogenesis by comparing cardiac gene expression in the presence and absence of the TGF-ß inhibitors. We found that the inhibitory effect of canonical Wnt/ß-catenin is sufficient to cause proper cardiomyocyte differentiation, but the TGF-ß pathway plays a vital role in enhancing the expression of the cardiomyocyte-specific marker (cTnT). It was found that quercetin, a p38MAPK inhibitor with the high energy dock to the active pocket of Wnt receptors, promotes cardiac differentiation via the inhibition of both Wnt and non-Smad TGF-ß pathways. Altogether, data presented here can contribute to the development of a feasible and efficient cardiac differentiation protocol as an "off-the-shelf" therapeutic source using novel natural agents for cardiac repair or regeneration.