3D bio scaffold support osteogenic differentiation of mesenchymal stem cells.

The regeneration of osteochondral lesions by tissue engineering techniques is challenging due to the lack of physicochemical characteristics and dual-lineage (osteogenesis and chondrogenesis). A scaffold with better mechanical properties and dual lineage capability is required for the regeneration of osteochondral defects. In this study, a hydrogel prepared from decellularized human umbilical cord tissue was developed and evaluated for osteochondral regeneration. Mesenchymal stem cells (MSCs) isolated from the umbilical cord were seeded with hydrogel for 28 days, and cell-hydrogel composites were cultured in basal and osteogenic media. Alizarin red staining, quantitative polymerase chain reaction, and immunofluorescent staining were used to confirm that the hydrogel was biocompatible and capable of inducing osteogenic differentiation in umbilical cord-derived MSCs. The findings demonstrate that human MSCs differentiated into an osteogenic lineage following 28 days of cultivation in basal and osteoinductive media. The expression was higher in the cell-hydrogel composites cultured in osteoinductive media, as evidenced by increased levels of messenger RNA and protein expression of osteogenic markers as compared to basal media cultured cell-hydrogel composites. Additionally, calcium deposits were also observed, which provide additional evidence of osteogenic differentiation. The findings demonstrate that the hydrogel is biocompatible with MSCs and possesses osteoinductive capability in vitro. It may be potentially useful for osteochondral regeneration.

Human umbilical cord-derived mesenchymal stem cells and their chondroprogenitor derivatives reduced pain and inflammation signaling and promote regeneration in a rat intervertebral disc degeneration model.

Intervertebral disc (IVD) degeneration is an asymptomatic pathophysiological condition and a strong causative factor of low back pain. There is no cure available except spinal fusion and pain management. Stem cell-based regenerative medicine is being considered as an alternative approach to treat disc diseases. The current study aimed to differentiate human umbilical cord-mesenchymal stem cells (hUC-MSCs) into chondrocyte-like cells and to elucidate their feasibility and efficacy in the degenerated IVD rat model. Chondrogenic induction medium was used to differentiate hUC-MSCs into chondroprogenitors. Rat tail IVD model was established with three consecutive coccygeal discs. qPCR was performed to quantify the molecular markers of pain and inflammation. Histological staining was performed to evaluate the degree of regeneration. Induced chondroprogenitors showed the expression of chondrogenic genes, SOX9, TGF-ß1, ACAN, BMP2, and GDF5. Immunocytochemical staining showed positive expression of chondrogenic proteins SOX9, TGF-ß1, TGF-ß2, and Collagen 2. In in vivo study, transplanted chondroprogenitors showed better survival, homing, and distribution in IVD as compared to normal MSCs. Expression of pain and inflammatory genes at day 5 of cell transplantation modulated immune response significantly. The transplanted labeled MSCs and induced chondroprogenitors differentiated into functional nucleus pulposus (NP) cells as evident from co-localization of red (DiI) and green fluorescence for SOX9, TGF-ß1, and TGF-ß2. Alcian blue and H & E staining showed standard histological features, indicating better preservation of the NP structure and cellularity than degenerated discs. hUC-MSCs-derived chondroprogenitors showed better regeneration potential as compared to normal MSCs. The pain and inflammation genes were downregulated in the treated group as compared to the degenerated IVD.

Identifying a C-terminal ATP binding sites-based novel Hsp90-Inhibitor in silico: a plausible therapeutic approach in Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is a progressive brain disorder, which gradually and irreversibly destroys the intellectual and cognitive abilities of the brain. Heat shock protein 90 (Hsp90α) is a molecular chaperone which was found to regulate the function of number of client proteins including tau that is involved in the cause of the AD. Inhibition of Hsp90α by C-Terminal domain (CTD) ATP binding-site blockage might be used as an effective treatment strategy against the disease via degradation of tau proteins that are involved in the progression of the disease. Till date, a variety of drugs have been identified as Hsp90α inhibitors, which include Novobiocin, Clorobiocin, Epigallocatechingallate (EGCG) and Derrubone. However, which drug among the four binds to the CTD ATP binding site strongly and what are the specific residue responsible for such binding, have not been reported so far. HYPOTHESIS: We hypothesize that binding site for ATP of Hsp90α CTD contains multiple ATP binding sites. We also hypothesize that a drug which can bind to the ATP binding site of CTD strongly can inhibit Hsp90α function which is in turn redirects towards the proteasomal degradation of diseased client protein like tau in AD. Such inhibition will find a novel therapeutic approach in the treatment of AD. EXPERIMENTAL DESIGN: The identification of ATP binding site of Hsp90α CTD was done using various software tools like Hex 6.3, CastP, protein Hydrophobicity plots, ATPint and LigPlot+ v.1.4.5. Docking experiments were conducted between Hsp90αCTD and its inhibitors at these ATP binding site using the Autodock 4.0. The docking energies were further compared to obtain the most effective Hsp90α inhibitor of CTD. RESULTS: From our experiments, Leucine (Leu) 665, Leu 666 and Leu 694 were predicted to be located in CTD ATP binding site. Furthermore, docking studies were performed of various Hsp90α inhibitors like Novobiocin, Clorobiocin, Epigallocatechingallate (EGCG) and Derrubone with the previously recognized ATP binding residues of CTD i.e. Leu 665, Leu 666 and Leu 694. The docking results of Derrubone showed the highest binding energy at all the three sites of ATP interaction. Additionally, Derrubone showed the best binding energy at Leu 666 (-7.53kcal/mol) compared to Leu 665 (-7.20kcal/mol) and Leu 694 (-6.67kcal/mol). CONCLUSION: Based on our findings, we propose that the recognized sites i.e. Leu665, Leu 666 and Leu694 could possibly be the binding sites of Hsp90α CTD for ATP and the Hsp90 inhibitors. It was predicted that Derrubone could bind with CTD of Hsp90α strongly and resulted tau protein degradation which might be considered to be a therapeutic approach in AD.