Evaluating potential of tissue-engineered cryogels and chondrocyte derived exosomes in articular cartilage repair.

Treatment of articular cartilage injuries especially osteochondral tissue requires intervention of bioengineered scaffold. In this study, we investigated the potential of the tissue-engineered cryogel scaffold fabricated using cryogelation technology. Two types of cryogels viz. chitosan-gelatin-chondroitin sulfate (CGC) for articular cartilage and nano-hydroxyapatite-gelatin (HG) for subchondral bone were fabricated. Further, novel bilayer cryogel designed using single process fabrication of two layers (CGC as top layer and HG as the lower layer) was designed to mimic osteochondral unit. CGC cryogel was tested for their biocompatibility using the enzymatically isolated chondrcoytes from goat articular cartilage while HG cryogel was tested using pre-osteoblast cell line. Extracellular vesicles, specifically exosomes were isolated from the spent media of chondrocytes to validate their effect over cell proliferation and migration which are required for defect healing and infiltration respectively. These isolated exosomes were characterized and analyzed for confirming their size distribution profile and visualized morphologically using advanced microscopy techniques. For cartilage part, CGC cryogels were examined as delivery system for delivering exosomes at defect site, where 80% of release was observed in 72 h. Release of 18.7 µg chondroitin sulfate/mg cryogel was obtained in a period of one week from CGC cryogel (termed cryogel extract) which has chondroprotective effect. Further, effect of exosome concentration (10 and 20 µg/ml), CGC extract and combination of exosome and CGC extract (Exo-Ex) were assessed over the chondrocytes. In addition, in vitro scratch wound assay was performed to analyse the migration capacity over the micro-injury when treated with exosomes, cryogel extract and Exo-Ex. The overall results thus answer key questions of therapeutic potential of chondrocyte exosomes, cryogel extract in addition to potential of CGC and HG cryogel for osteochondral repair.

Antioxidant and Trilayered Electrospun Small-Diameter Vascular Grafts Maintain Patency and Promote Endothelialisation in Rat Femoral Artery.

Vascular grafts with a small diameter encounter inadequate patency as a result of intimal hyperplasia development. In the current study, trilayered electrospun small-diameter vascular grafts (PU-PGACL + GA) were fabricated using a poly(glycolic acid) and poly(caprolactone) blend as the middle layer and antioxidant polyurethane with gallic acid as the innermost and outermost layers. The scaffolds exhibited good biocompatibility and mechanical properties, as evidenced by their 6 MPa elastic modulus, 4 N suture retention strength, and 2500 mmHg burst pressure. Additionally, these electrospun grafts attenuated cellular oxidative stress and demonstrated minimal hemolysis (less than 1%). As a proof-of-concept, the preclinical evaluation of the grafts was carried out in the femoral artery of rodents, where the conduits demonstrated satisfactory patency. After 35 days of implantation, ultrasound imaging depicted adequate blood flow through the grafts, and the computed vessel diameter and histological staining showed no significant stenosis issue. Immunohistochemical analysis confirmed matrix deposition (38% collagen I and 16% elastin) and cell infiltration (42% for endothelial cells and 55% for smooth muscle cells) in the explanted grafts. Therefore, PU-PGACL + GA showed characteristics of a clinically relevant small-diameter vascular graft, facilitating re-endothelialization while preserving the anticoagulant properties of the synthetic blood vessels.

Ameliorating impaired cardiac function in myocardial infarction using exosome-loaded gallic-acid-containing polyurethane scaffolds.

Myocardial infarction (MI) can be tackled by implanting cardiac patches which provide mechanical support to the heart. However, most tissue-engineered scaffolds face difficulty in attenuating oxidative stress, maintaining mechanical stability, and regenerating damaged cardiomyocytes. Here, we fabricated elastic cryogels using polyurethane modified with antioxidant gallic acid in its backbone (PUGA) and further coated them with decellularized extracellular matrix (dECM) to improve adhesiveness, biocompatibility and hemocompatibility. The scaffold was functionalized with exosomes (EXO) isolated from adipose-derived stem cells having regenerative potential. PUGA-dECM + EXO was tested in a rat model with induced MI where echocardiography after 8 weeks of implantation showed significant recovery in treatment group. Histological analysis revealed a decrease in fibrosis after application of patch and promotion of angiogenesis with reduced oxidative stress was shown by immunostaining. Expression of cardiac tissue contractile function marker was also observed in treatment groups. Thus, the proposed biomaterial has a promising application to be utilized as a patch for cardiac regeneration. More detailed studies with larger animal species are needed for using these observations for specific applications.

Preparation of thermo-responsive polymer encapsulated exosomes and its role as a therapeutic agent for blood clot lysis.

Tissue plasminogen activators induce enzymatic activation of plasminogen to plasmin that cleaves fibrin strands in blood clots. In the present study, extracellular vesicles such as exosomes from fibrosarcoma cell line HT1080 were utilized as clot-busting agents. These exosomes were being used for clot lysis of whole blood which showed 28% lysis within 10 h, which was comparable to that of the streptokinase (commercial plasmin activator) with no significant difference. These exosomes were able to facilitate the migration of endothelial cells in a scratch wound assay where normalized wound area remaining was 7.5% at 18 h. Also, exosomes aided in attenuation of oxidative stress generated on the cells, thereby maintaining cell viability. These exosomes were further encapsulated in a thermo-responsive polymer for better localized delivery that showed no cytotoxic effects, and sustained delivery was achieved up to a concentration of 117 µg/mL in 25 days, which corresponds to around 65% of the total amount of exosomes added. When a combination of exosomes and thermo-responsive polymer was utilized, the clot lysis activity reached to around 22% in 72 h. Thus, it proves the potential of this combinatorial approach which can be effectively used for thrombus degradation and healing of endothelium lining in damaged blood vessels.

Comparative analysis of secondary metabolite gene clusters in different strains of Magnaporthe oryzae.

Rice blast caused by Magnaporthe oryzae continues to be a major constraint in rice production worldwide. Rice is one of the staple crops in India and rice blast causes huge economic losses. Interestingly, the Indian subcontinent is the centre for origin and diversity of rice as well as the Magnaporthe species complex. Secondary metabolites are known to play important role in pathogenesis and M. oryzae has high potential of genes involved in secondary metabolism but, unfortunately most of them remain uncharacterized. In the present study, we analysed the draft genome assemblies of M. oryzae strains isolated from different parts of India, for putative secondary metabolite key gene (SMKG) clusters encoding polyketide synthases, non-ribosomal peptide synthetases, diterpene cyclases and dimethylallyl tryptophan synthase. Based on the complete genome sequence of 70-15 strain and its previous reports of identified SMKGs, we have identified the key genes for the interrogated strains. Expression analysis of these genes amongst different strains indicates how they have evolved depending on the host and environmental conditions. To our knowledge, this study is first of its kind where the secondary metabolism genes and their role in functional adaptation were studied across several strains of M. oryzae.