Santosh Mathapati

Biomimetic acellular detoxified glutaraldehyde cross-linked bovine pericardium for tissue engineering.

Glutaraldehyde (GLUT) processing, cellular antigens, calcium ions in circulation, and phospholipids present in the native tissue are predominantly responsible for calcification, degeneration, and lack of natural microenvironment for host progenitor cell migration in tissue implants. The study presents an improved methodology for adhesion and proliferation of endothelial progenitor cells (EPCs) without significant changes in biomechanical and biodegradation properties of the processed acellular bovine pericardium. The anti-calcification potential of the processed tissue was enhanced by detoxification of GLUT-cross-linked bovine pericardium by decellularization, pretreating it with ethanol or removing the free aldehydes by citric acid treatment and lyophilization. The treated tissues were assessed for biomechanical properties, GLUT ligand quantification, adhesion, proliferation of EPCs, and biodegradability. The results indicate that there was no significant change in biomechanical properties and biodegradability when enzymatic hydrolysis (p>0.05) is employed in detoxified acellular GLUT cross-linked tissue (DBP-G-CA-ET), compared with the native detoxified GLUT cross-linked bovine pericardium (NBP-G-CA-ET). DBP-G-CA-ET exhibited a significant (p>0.05) increase in the viability of EPCs and cell adhesion as compared to acellular GLUT cross-linked bovine pericardium (p<0.05). Lyophilized acellular detoxified GLUT cross-linked bovine pericardium, employed in our study as an alternative to conventional GLUT cross-linked bovine pericardium, might provide longer durability and better biocompatibility, and reduce calcification. The developed bovine pericardium patches could be used in cardiac reconstruction and repair, arteriotomy, soft tissue repair, and general surgical procedures with tissue regeneration dimensions.

Inflammatory responses of tissue-engineered xenografts in a clinical scenario.

Acellular tissue-engineered (ATE) xenografts and homografts are used in clinical cardiovascular surgery. The present study examined the specific role of carbohydrate antigen (α-Gal and T-antigen) in immune response after decellularisation in tissue-engineered xenografts (porcine pulmonary artery and bovine jugular vein). An enzyme-linked immunosorbent assay (ELISA) was used to ascertain whether implantation of bioprostheses, ATE xenografts and mechanical valve replacement result in augmentation of anti-α-Gal IgM antibodies within eight days of surgery (each group, n=6). Kinetics of host inflammatory response on surgically explanted ATE xenografts was also studied. Immunostaining for α-Gal and T-antigen detected the presence of them in the native tissue but they were absent in processed ATE xenografts from the same tissue. A significant increase in the concentration of anti-α-Gal IgM antibodies was observed in the serum of bioprosthetic valve recipients as compared to ATE xenograft recipients (P<0.05). Organised collagen, and decreased inflammatory response with increase in endothelisation and vascularisation was evident beyond one year of surgery as compared to early periods in ATE xenografts. This study demonstrates that decellularisation of xenografts and further processing of these tissues enabled reduction of inflammatory stimulus with autologous recellularisation with no calcification.

In Vitro Hepatic Trans-Differentiation of Human Mesenchymal Stem Cells Using Sera from Congestive/Ischemic Liver during Cardiac Failure

Cellular therapy for end-stage liver failures using human mesenchymal stem cells (hMSCs)-derived hepatocytes is a potential alternative to liver transplantation. Hepatic trans-differentiation of hMSCs is routinely accomplished by induction with commercially available recombinant growth factors, which is of limited clinical applications. In the present study, we have evaluated the potential of sera from cardiac-failure-associated congestive/ischemic liver patients for hepatic trans-differentiation of hMSCs. Results from such experiments were confirmed through morphological changes and expression of hepatocyte-specific markers at molecular and cellular level. Furthermore, the process of mesenchymal-to-epithelial transition during hepatic trans-differentiation of hMSCs was confirmed by elevated expression of E-Cadherin and down-regulation of Snail. The functionality of hMSCs-derived hepatocytes was validated by various liver function tests such as albumin synthesis, urea release, glycogen accumulation and presence of a drug inducible cytochrome P450 system. Based on these findings, we conclude that sera from congestive/ischemic liver during cardiac failure support a liver specific microenvironment for effective hepatic trans-differentiation of hMSCs in vitro.

A Patient-Inspired Ex Vivo Liver Tissue Engineering Approach with Autologous Mesenchymal Stem Cells and Hepatogenic Serum.

Design and development of ex vivo bioengineered liver tissue substitutes intended for subsequent in vivo implantation has been considered therapeutically relevant to treat many liver diseases that require whole-organ replacement on a long-term basis. The present study focus on patient-inspired ex vivo liver tissue engineering strategy to generate hepatocyte-scaffold composite by combining bone marrow mesenchymal stem cells (BMSCs) derived from cardiac failure patients with secondary hyperbilirubinemia as primers of hepatic differentiation and hepatocyte growth factor (HGF)-enriched sera from same individuals as hepatic inducer. A biodegradable and implantable electrospun fibrous mesh of poly-l-lactic acid (PLLA) and gelatin is used as supporting matrix (average fiber diameter = 285 ± 64 nm, porosity = 81 ± 4%, and average pore size = 1.65 ± 0.77 μm). The fibrous mesh supports adhesion, proliferation, and hepatic commitment of patient-derived BMSCs of adequate stemness using HGF-enriched sera generating metabolically competent hepatocyte-like cells, which is comparable to the hepatic induction with defined recombinant growth factor cocktail. The observed results confirm the combinatorial effects of nanofiber topography and biochemical cues in guiding hepatic specification of BMSCs. The fibrous mesh-hepatocyte construct developed in this study using natural growth factors and BMSCs of same individual is promising for future therapeutic applications in treating damaged livers.

High cholesterol diet increases MMP9 and CD40 immunopositivity in early atherosclerotic plaque in rabbits.

Inflammation is one of the important contributing factors for the development of atherosclerosis and heart disease. Inflammation leads to the mobilization of various cells in developing atherosclerotic plaque and simultaneously triggers the up-regulation of various cytokine secretions from effector cells. To understand early molecular events during atherosclerosis we developed a rabbit model in which male New Zealand White rabbits were fed high cholesterol diets for 12 weeks. Total cholesterol (TC), triglycerides (TG), low density lipoprotein-cholesterol (LDL-C) and high sensitivity C-reactive protein (hs-CRP) were significantly increased in the high cholesterol diet group when compared to the control group during the experimental period (P<0.05). In parallel, the immunolocalization of CD40, MMP9, S100, CD68, α-smooth muscle actin and von Willebrand factor (vWF) in developing atherosclerotic plaque of the aorta and carotid artery was increased in comparison with the controls fed with a regular diet (P<0.05). From the present study, we conclude that a high cholesterol diet up-regulates CD68 and CD40, which may play a possible role in the remodelling and destabilization of the atherosclerotic plaque of arteries with the up-regulation of MMP9 and hsCRP.

Crosslinked acellular saphenous vein for small-diameter vascular graft.

Objective: Patients with congenital and acquired heart diseases or arteriopathy require small-diameter vascular grafts for arterial reconstruction. Autologous veins are the most suitable graft, but when absent, an alternative is necessary. This work addresses the issue. Background: Tissue-engineering efforts to create such grafts by modifications of acellular natural scaffolds are considered a promising area. Methods: Homologous saphenous veins harvested from cadavers and organ donors were processed by decellularization with detergent and enzymatic digestion, followed by crosslinking by dye-mediated photooxidation. They were validated for acellularity, mechanical strength, and crosslink stability. In-vitro and in-vivo cytotoxicity and hemocompatibility studies were conducted. Collagen conformity was studied by Fourier transform infrared spectroscopy, and heat stability by differential scanning calorimetry. A limited large animal study was performed. Results: The processing method delivered biocompatible, hemocompatible, effectively crosslinked grafts, with high heat stability of 126 ℃, an enthalpy value of 183.5 J·g−1, and collagen conformity close to that of the native vein. The mechanical strength was 250% better than the native vein. The presence of extracellular matrix proteins allowed the acellular vein to become a triple-layered vascular structure in the sheep venous system. Conclusion: Crosslinking after decellularization by the dye-mediated photooxidation method could be reproduced in any human vein to obtain a small-diameter vascular grafts.

Nanofiber-reinforced myocardial tissue-construct as ventricular assist device.

Objectives This study aimed to create a myocardial tissue construct by tissue engineering to repair, replace, and regenerate damaged cardiac tissue. Methods and results Human cardiac muscles harvested from a homograft heart retrieval system were decellularized followed by coating with electrospun nanofibers to make them amenable to scaffolding. These processed cardiac tissues were nourished in modified media having ischemic cardiac tissue conditioned media in 6 separate experimental variants, and cord blood mononuclear cells were injected into 4 of them. On the 17th day of culture, the nanofiber-coated scaffolds injected with mononuclear cells and/or reinforced by electrical and mechanical forces, started contracting spontaneously at varying rates, while the control remain noncontractile. Histological staining confirmed the pre-culture acellularity as well as post-culture stem cell viability, and revealed expression of troponin I and cardiac myosin. The acellular processed scaffold when implanted into sheep ischemic myocardial apex revealed transformation into sheep myocardium after 4 months of implantation. Conclusion These results provide direct evidence for the re-cellularization of decellularized cardiac tissue grafts reinforced with a polymer nanofiber coating, by human mononuclear cells injection, leading to generation of a tissue-engineered myocardial construct.