Fabrication of a Smart Fibrous Biomaterial That Harbors an Active TGF-ß1 Peptide: A Promising Approach for Cartilage Regeneration.

The regeneration of articular cartilage remains a serious problem in various pathological conditions such as osteoarthritis, due to the tissue's low self-healing capacity. The latest therapeutic approaches focus on the construction of biomaterials that induce cartilage repair. This research describes the design, synthesis, and investigation of a safe, "smart", fibrous scaffold containing a genetically incorporated active peptide for chondrogenic induction. While possessing specific sequences and the respective mechanical properties from natural fibrous proteins, the fibers also incorporate a Transforming Growth Factor-ß1 (TGF-ß1)-derived peptide (YYVGRKPK) that can promote chondrogenesis. The scaffold formed stable porous networks with shear-thinning properties at 37 °C, as shown by SEM imaging and rheological characterization, and were proven to be non-toxic to human dental pulp stem cells (hDPSCs). Its chondrogenic capacity was evidenced by a strong increase in the expression of specific chondrogenesis gene markers SOX9, COL2, ACAN, TGFBR1A, and TGFBR2 in cells cultured on "scaffold-TGFß1" for 21 days and by increased phosphorylation of intracellular signaling proteins Smad-2 and Erk-1/2. Additionally, intense staining of glycosaminoglycans was observed in these cells. According to our results, "scaffold-TGFß1" is proposed for clinical studies as a safe, injectable treatment for cartilage degeneration.

Inhibition of PERK Kinase, an Orchestrator of the Unfolded Protein Response (UPR), Significantly Reduces Apoptosis and Inflammation of Lung Epithelial Cells Triggered by SARS-CoV-2 ORF3a Protein.

SARS-CoV-2 ORF3a accessory protein was found to be involved in virus release, immunomodulation and exhibited a pro-apoptotic character. In order to unravel a potential ORF3a-induced apoptotic and inflammatory death mechanism, lung epithelial cells (A549) were transfected with in vitro synthesized ORF3a mRNA. The protein's dynamic involvement as "stress factor" for the endoplasmic reticulum, causing the activation of PERK kinase and other UPR-involved proteins and therefore the upregulation of their signaling pathway executioners (ATF6, XBP-1s, PERK, phospho eIF2a, ATF4, CHOP, GADD34), has been clearly demonstrated. Furthermore, the overexpression of BAX and BH3-only pro-apoptotic protein PUMA, the upregulation of Bcl-2 family genes (BAX, BAK, BID, BAD), the reduced expression of Bcl-2 in mRNA and protein levels, and lastly, the cleavage of PARP-1 and caspase family members (caspase-3,-8 and -9) indicate that ORF3a displays its apoptotic character through the mitochondrial pathway of apoptosis. Moreover, the upregulation of NFκB, phosphorylation of p65 and IκΒα and the elevated expression of pro-inflammatory cytokines (IL-1b, IL-6, IL-8 and IL-18) in transfected cells with ORF3a mRNA indicate that this protein causes the inflammatory response through NFκB activation and therefore triggers lung injury. An intriguing finding of our study is that upon treatment of the ORF3a-transfected cells with GSK2606414, a selective PERK inhibitor, both complications (apoptosis and inflammatory response) were neutralized, and cell survival was favored, whereas treatment of transfected cells with z-VAD (a pan-caspase inhibitor) despite inhibiting cell death, could not ameliorate the inflammatory response of transfected A549 cells. Given the above, we point out that PERK kinase is a "master tactician" and its activation constitutes the main stimulus for the emergence of ORF3a apoptotic and inflammatory nature and therefore could serve as potential target for developing novel therapeutic approaches against COVID-19.

Enhancement of mesenchymal stem cells' chondrogenic potential by type II collagen-based bioscaffolds.

BACKGROUND: Osteoarthritis (OA) is a common degenerative chronic disease accounting for physical pain, tissue stiffness and mobility restriction. Current therapeutic approaches fail to prevent the progression of the disease considering the limited knowledge on OA pathobiology. During OA progression, the extracellular matrix (ECM) of the cartilage is aberrantly remodeled by chondrocytes. Chondrocytes, being the main cell population of the cartilage, participate in cartilage regeneration process. To this end, modern tissue engineering strategies involve the recruitment of mesenchymal stem cells (MSCs) due to their regenerative capacity as to promote chondrocyte self-regeneration. METHODS AND RESULTS: In the present study, we evaluated the role of type II collagen, as the main matrix macromolecule in the cartilage matrix, to promote chondrogenic differentiation in two MSC in vitro culture systems. The chondrogenic differentiation of human Wharton's jelly- and dental pulp-derived MSCs was investigated over a 24-day culture period on type II collagen coating to improve the binding affinity of MSCs. Functional assays, demonstrated that type II collagen promoted chondrogenic differentiation in both MSCs tested, which was confirmed through gene and protein analysis of major chondrogenic markers. CONCLUSIONS: Our data support that type II collagen contributes as a natural bioscaffold enhancing chondrogenesis in both MSC models, thus enhancing the commitment of MSC-based therapeutic approaches in regenerative medicine to target OA and bring therapy closer to the clinical use.

A Novel Drastic Peptide Genetically Adapted to Biomimetic Scaffolds "Delivers" Osteogenic Signals to Human Mesenchymal Stem Cells.

This work describes the design, preparation, and deep investigation of "intelligent nanobiomaterials" that fulfill the safety rules and aim to serve as "signal deliverers" for osteogenesis, harboring a specific peptide that promotes and enhances osteogenesis at the end of their hydrogel fibers. The de novo synthesized protein fibers, besides their mechanical properties owed to their protein constituents from elastin, silk fibroin and mussel-foot adhesive protein-1 as well as to cell-attachment peptides from extracellular matrix glycoproteins, incorporate the Bone Morphogenetic Protein-2 (BMP2) peptide (AISMLYLDEN) that, according to our studies, serves as "signal deliverer" for osteogenesis. The osteogenetic capacity of the biomaterial has been evidenced by investigating the osteogenic marker genes ALP, RUNX2, Osteocalcin, COL1A1, BMPR1A, and BMPR2, which were increased drastically in cells cultured on scaffold-BMP2 for 21 days, even in the absence of osteogenesis medium. In addition, the induction of phosphorylation of intracellular Smad-1/5 and Erk-1/2 proteins clearly supported the osteogenetic capacity of the biomaterial.

De Novo Synthesis of Elastin-like Polypeptides (ELPs): An Applied Overview on the Current Experimental Techniques.

Elastin-like polypeptides (ELPs) are protein-based biopolymers genetically produced from polypeptides composed of a repeating pentapeptide sequence V-P-G-X-G. The inherent properties of recombinant ELPs, such as smart nature, controlled sequence complexity, physicochemical properties, and biocompatibility, make these polymers suitable for use in nanobiotechnological applications, as biofunctionalized scaffolds for tissue-engineering purposes and drug delivery. In this work, we report the design and synthesis of two elastomeric self-assembling polypeptides (ELPs) that mimic the endogenous human tropoelastin. Using molecular biology techniques, two artificial genes that encode two ELP concatemers of approximate molecular mass 60 kDa, one of them carrying biotin-binding peptide motifs, were constructed. These motifs could facilitate biofunctionalization of the ELPs through tethering biotinylated factors, such as growth factors. The ELPs were heterologously overexpressed in E. coli and subsequently purified in two steps: a nonchromatographic technique by organic solvent extraction, followed by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. The characterization of the biochemical properties and biocompatibility of ELPs was also performed in this study. The ELP carrying the biotin-binding motifs was tested for its capability to bind biotin, and indeed, it was observed that it can bind biotinylated proteins specifically. Additionally, results concerning the cytotoxicity of the ELPs exhibited excellent compatibility of the ELPs with mammalian cells in vitro. We anticipate that these ELPs can be used as components of a scaffold that mimics the extracellular matrix (ECM) for the regeneration of endogenously highly elastic tissues.

Effect of a Bone Morphogenetic Protein-2-derived peptide on the expression of tumor marker ZNF217 in osteoblasts and MCF-7 cells.

Zinc Finger Protein 217 (ZNF217), a transcription factor and oncogene product, has been found to dysregulate Bone Morphogenetic Protein (BMP) signaling and induce invasion in breast tumors. In this study, the effect of BMP-2 or an active BMP-2 peptide, AISMLYLDEN, on the expression of ZNF217, BMP4 and CDK-inhibitor p21 gene, CDKN1A, was investigated in MCF-7 breast cancer cells. In parallel, the entire protein (BMP-2) as well as the aforementioned peptide were investigated in hDPSCs during osteogenic differentiation. The treatment of MCF-7 cancer cells with different concentrations of peptide AISMLYLDEN showed that the addition of 22.6 ng/ml was more effective in comparison to the other used concentrations. In particular, 48 h after treatment, CDKN1A and BMP4 mRNA levels were substantially increased in contrast to ZNF217 mRNA levels which were decreased. These results are strongly supported by BrdU assay that clearly indicated inhibition of cancer cell proliferation. Taken together, these results open ways for a concurrent use, at appropriate concentrations, of the peptide AISMLYLDEN during conventional therapeutic treatment in breast tumors with a metastatic tendency to the bones. Regarding the effect of the entire protein as well as its peptide on hDPSCs differentiation into osteocytes, the mRNA levels of osteocalcin, an osteogenic marker, showed that the peptide enhanced osteogenesis at a higher degree in comparison to the entire BMP-2 without however altering ZNF217, CDKN1A and BMP4 expression levels, which remained as expected of non-cancer cells.

Regulation of expression of the p21CIP1 gene by the transcription factor ZNF217 and MDM2.

Using mouse double minute 2 (MDM2) protein-specific affinity chromatography and mass spectrometry, we have isolated the protein product of the oncogene znf217, which is a transcription factor and a component of a Hela-S-derived HDAC1 complex, as a novel MDM2-interacting protein. When co-expressed in cultured cancer cells, ZNF217 forms a complex with MDM2 and its ectopic over-expression reduces the steady-state levels of acetylated p53 in cell lines, suppressing its ability to activate the expression of a p21 promoter construct. In-silico analysis of the p21 promoter revealed the presence of several ZNF217-binding sites. These findings suggest that MDM2 controls p21 expression by at least 2 mechanisms: through ZNF217-mediated recruitment of HDAC1/MDM2 activity, which inhibits p53 acetylation; and through direct interaction with its binding site(s) on the p21 promoter.