In Vivo Wound-Healing Efficacy of Insulin-Loaded Strontium-Cross-Linked Alginate-Based Hydrogels in Diabetic Rats.

The wound-healing effect of insulin is well studied and reported. However, prolonged topical application of insulin without compromising its biological activity is still a challenge. In this study, the effect of topically delivered insulin on promoting wound healing in diabetic animals was evaluated. Alginate diamine PEG-g-poly(PEGMA) (ADPM2S2) was the material used for the topical delivery of insulin. ADPM2S2 hydrogels release insulin and strontium ions, and they synergistically act to regulate different phases of wound healing. Insulin was released from the ADPM2S2 hydrogel for a period of 48 h, maintaining its structural stability and biological activity. In vitro studies were performed under high-glucose conditions to evaluate the wound-healing potential of insulin. Insulin-loaded ADPM2S2 hydrogels showed significant improvement in cell migration, proliferation, and collagen deposition, compared to control cells under high-glucose conditions. Immunostaining studies in L929 cells showed a reduction in phospho Akt expression under high-glucose conditions, and in the presence of insulin, the expression increased. The gene expression studies revealed that insulin plays an important role in regulating the inflammatory phase and macrophage polarization, which favors accelerated wound closure. In vivo experiments in diabetic rat excision wounds treated with insulin-loaded ADPM2S2 showed 95% wound closure within 14 days compared with 82% in control groups. Thus, both the in vitro and in vivo results signify the therapeutic potential of topically delivered insulin in wound management under high-glucose conditions.

Mitigation of Fibrosis after Myocardial Infarction in Rats by Using a Porcine Cholecyst Extracellular Matrix.

Fibrosis that occurs after nonfatal myocardial infarction (MI) is an irreversible reparative cardiac tissue remodeling process characterized by progressive deposition of highly cross-linked type I collagen. No currently available therapeutic strategy prevents or reverses MI-associated fibrotic scarring of myocardium. In this study, we used an epicardial graft prepared of porcine cholecystic extracellular matrix to treat experimental nonfatal MI in rats. Graft-assisted healing was characterized by reduced fibrosis, with scanty deposition of type I collagen. Histologically, the tissue response was associated with a favorable regenerative reaction predominated by CD4-positive helper T lymphocytes, enhanced angiogenesis, and infiltration of proliferating cells. These observations indicate that porcine cholecystic extracellular matrix delayed the fibrotic reaction and support its use as a potential biomaterial for mitigating fibrosis after MI. Delaying the progression of cardiac tissue remodeling may widen the therapeutic window for management of scarring after MI.

A porcine cholecystic extracellular matrix conductive scaffold for cardiac tissue repair.

Cardiac tissue engineering using cells, scaffolds or signaling molecules is a promising approach for replacement or repair of damaged myocardium. This study addressed the contemporary need for a conductive biomimetic nanocomposite scaffold for cardiac tissue engineering by examining the use of a gold nanoparticle-incorporated porcine cholecystic extracellular matrix for the same. The scaffold had an electrical conductivity (0.74 ± 0.03 S/m) within the range of native myocardium. It was a suitable substrate for the growth and differentiation of cardiomyoblast (H9c2) as well as rat mesenchymal stem cells to cardiomyocyte-like cells. Moreover, as an epicardial patch, the scaffold promoted neovascularisation and cell proliferation in infarcted myocardium of rats. It was concluded that the gold nanoparticle coated cholecystic extracellular matrix is a prospective biomaterial for cardiac tissue engineering.

Surface Modification of Polypropylene Mesh with a Porcine Cholecystic Extracellular Matrix Hydrogel for Mitigating Host Tissue Reaction.

Polypropylene (PP) meshes are widely used for repairing skeletal muscle defects like abdominal hernia despite the chances of undesirable pro-inflammatory tissue reactions that demand revision surgeries in about 45% of cases. Attempts have been made to address the problem by modifying the mesh surface and architecture. These procedures have yielded only incremental improvements in the management of overall postoperative complications, and the search for a clinically viable therapeutic strategy continues. This study deployed a tissue engineering approach for mitigating PP-induced adverse tissue reaction by dip-coating the mesh with a hydrogel formulation of the porcine cholecystic extracellular matrix (CECM). The biomaterial properties of the CECM hydrogel-coated PP (C-PP) meshes were studied and their biocompatibility was evaluated by in vitro and in vivo tests based on ISO standards. Further, the nature of tissue reactions induced by the hydrogel-coated mesh and a commercial PP hernia repair graft was compared in a rat model of partial-thickness abdominal wall defect. Histomorphologically, in comparison with the PP graft-induced tissue reaction, C-PP caused a favorable graft-acceptance response characterized by reduced numbers of pro-inflammatory M1 macrophages and cytotoxic lymphocytes. Remarkably, the differential inflammatory response of the C-PP graft-assisted healing was associated with a fibrotic reaction predominated by deposition of type I collagen rather than type III collagen, as desired during skeletal muscle repair. It was concluded that the CECM hydrogel is a potential biomaterial for surface modification of polymeric biomedical devices.

Gelatin-Modified Cholecyst-Derived Scaffold Promotes Angiogenesis and Faster Healing of Diabetic Wounds.

Compromised angiogenesis is a major factor contributing delayed wound healing in diabetic patients. Graft-assisted healing using synthetic and natural scaffolds supplemented with micromolecules for stimulating angiogenesis is the contemporary tissue engineering strategy for treating diabetic wounds. This study deployed the carbodiimide chemical reaction for coupling gelatin with a porcine cholecyst-derived scaffold (CDS) for enhancing angiogenesis. The modification was confirmed by the trinitrobenzene sulfonic acid assay and scanning electron microscopy. The gelatin-coupled CDS was more stable than the bare CDS in an in vitro proteolytic environment and allowed survival of keratinocytes (HaCaT), indicating its suitability for chronic skin wound application. The gelatin coupling brought significant improvement in the in vitro angiogenic potential of the CDS as evident from the enhanced viability of endothelial cells. An in ovo chorioallantoic membrane assay also demonstrated the angiogenic potential of the modified scaffold. Further, the modified scaffold promoted angiogenesis and aided faster healing of full-thickness excision wounds in streptozotocin-induced diabetic rats. It is concluded that the gelatin-coupled CDS is a potential advanced wound care material for treating diabetic wounds.

A cholecystic extracellular matrix-based hybrid hydrogel for skeletal muscle tissue engineering.

Tailoring the properties of extracellular matrix (ECM) based hydrogels by conjugating with synthetic polymers is an emerging method for designing hybridhydrogels for a wide range of tissue engineering applications. In this study, poly(ethylene glycol) diacrylate (PEGDA), a synthetic polymer at variable concentrations (ranging from 0.2 to 2% wt/vol) was conjugated with porcine cholecyst derived ECM (C-ECM) (1% wt/vol) and prepared a biosynthetic hydrogel having enhanced physico-mechanical properties, as required for skeletal muscle tissue engineering. The C-ECM was functionalized with acrylate groups using activated N-hydroxysuccinimide ester-based chemistry and then conjugated with PEGDA via free-radical polymerization in presence of ammonium persulfate and ascorbic acid. The physicochemical characteristics of the hydrogels were evaluated by Fourier transform infrared spectroscopy and environmental scanning electron microscopy. Further, the hydrogel properties were studied by evaluating rheology, swelling, gelation time, percentage gel fraction, in vitro degradation, and mechanical strength. Biocompatibility of the gel formulations were assessed using the C2C12 skeletal myoblast cells. The hydrogel formulations containing 0.2 and 0.5% wt/vol of PEGDA were non-cytotoxic and found suitable for growth and proliferation of skeletal myoblasts. The study demonstrated a method for modulating the properties of ECM hydrogels through conjugation with bio-inert polymers for skeletal muscle tissue engineering applications.

Hepatoprotective effect of Lobelia alsinoides Lam. in Wistar rats.

BACKGROUND: Traditional healing practitioners of South India use fine paste (an Ayurvedic dosage form known as 'kalka') of Lobelia alsinoides Lam., an ethno medicinal plant for curing hepatic diseases. OBJECTIVE: To evaluate in-vivo hepatoprotective effect of a candidate formulation viz. kalka containing whole plant (L. alsinoides Lam.) in rat model of Carbon-tetrachloride (CCl4) induced hepatotoxicity. MATERIALS & METHODS: Hepatotoxicity was induced in Wistar albino rats by oral administration of 1.25 ml/kg CCl4 once every day for 7 consecutive days. A candidate kalka formulation (fine paste) was prepared and administered to rats at different dose rates of 0.54 g/kg, 1.08 g/kg and 2.16 g/kg daily. At the end of the study-period, the serum levels of aspartate amino transferase (AST), alanine amino transferase (ALT), alkaline phosphatase (ALP), total bilirubin, total protein, albumin and total cholesterol were monitored. Further, the hepatic pathology was evaluated for assessing the extent of hepatotoxicity in the control and hepatoprotective effect in treatment groups. Meanwhile in-vitro antioxidant activity of kalka was evaluated by hydroxy radical, nitric oxide and DPPH (2, 2 diphenyl-1-picrylhydrazil) radical scavenging assays. Further, a 'limit test' was done in accordance with OECD Guidelines 425 (acute toxicity). RESULTS: The animals treated with the fine paste of L. alsinoides did not show an elevation in the biochemical values compared to CCl4 treated rats and during histomorphologic evaluation, hepatoprotective effect was evident with scattered mitotic figures in the parenchyma. Acute toxicity evaluation indicated that doses up to 2500 mg/kg are not toxic to rats. It has a good anti-oxidant activity also. CONCLUSIONS: From the study, it was obvious that L. alsinoides had significant hepatoprotective effect in CCl4 induced liver toxicity in rats. This ethno medicinal plant is certainly a promising hepatoprotective drug in liver disorders.

Controlled cross-linking of porcine cholecyst extracellular matrix for preparing tissue engineering scaffold.

Treatment with cross-linking agents for stabilizing biomolecules is an integral step during the preparation of many extracellular matrix-based tissue engineering scaffolds from mammalian organs. However, excess cross-linking may cause nonavailability of biomolecules and consequent deterioration of bioinductive properties of the scaffold. The present study considered controlling the extent of cross-linking in a porcine cholecyst extracellular matrix scaffold prepared by a nonenzymatic and nondetergent method, by ex situ incubation of the source organ in varying concentrations of neutral buffered formaldehyde (10, 4, 1 or 0%; v/v) for in situ cross-linking of biomolecules. Reduction of the formaldehyde concentration resulted in an increase in the extent of biodegradation and a decrease in the compactness of the mesh-like surface microarchitecture of the scaffold. Retention of collagen was maximum when treated with 10% neutral buffered formaldehyde without any variation in the content of elastin and sulphated glycosaminoglycans. Although there was a reduction in the quantity of growth factors following the cross-linking, fibroblasts remained viable on the scaffolds. The retention of major biomolecule was maximum and autodigestion was minimum in the scaffold prepared by the ex situ treatment of cholecyst in 10% neutral buffered formalin and found suitable for preparing the tissue engineering scaffold.

A gold nanoparticle coated porcine cholecyst-derived bioscaffold for cardiac tissue engineering.

Extracellular matrices of xenogeneic origin have been extensively used for biomedical applications, despite the possibility of heterogeneity in structure. Surface modification of biologically derived biomaterials using nanoparticles is an emerging strategy for improving topographical homogeneity when employing these scaffolds for sophisticated tissue engineering applications. Recently, as a tissue engineering scaffold, cholecyst derived extracellular matrix (C-ECM) has been shown to have several advantages over extracellular matrices derived from other organs such as jejunum and urinary bladder. This study explored the possibility of adding gold nanoparticles, which have a large surface area to volume ratio on C-ECM for achieving homogeneity in surface architecture, a requirement for cardiac tissue engineering. In the current study, gold nanoparticles (AuNPs) were synthesized and functionalised for conjugating with a porcine cholecystic extracellular matrix scaffold. The conjugation of nanoparticles to C-ECM was achieved by 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide/N-hydroxysuccinimide chemistry and further characterized by Fourier transform infrared spectroscopy, environmental scanning electron microscopy, energy dispersive X-ray spectroscopy and thermogravimetric analysis. The physical properties of the modified scaffold were similar to the original C-ECM. Biological properties were evaluated by using H9c2 cells, a cardiomyoblast cell line commonly used for cellular and molecular studies of cardiac cells. The modified scaffold was found to be a suitable substrate for the growth and proliferation of the cardiomyoblasts. Further, the non-cytotoxic nature of the modified scaffold was established by direct contact cytotoxicity testing and live/dead staining. Thus, the modified C-ECM appears to be a potential biomaterial for cardiac tissue engineering.

Comparative profiling of extractable proteins in extracellular matrices of porcine cholecyst and jejunum intended for preparation of tissue engineering scaffolds.

Scaffolds prepared from cholecyst and jejunum have differential immunological potential, despite similar biocompatibility, when used as subcutaneous grafts. The reason for differential immunogenicity is probably due to differences in the nature of protein composition and biomolecules in the extracellular matrices (ECMs) of source organs that are used for preparation of the scaffolds. Against this background, the present study aims to identify the extractable proteins of ECMs derived from porcine cholecyst and jejunum. The proteins were extracted and identified through a conventional database search following sodium dodecyl sulfate-polyacrylamide gel-electrophoresis separation and mass spectroscopy. The resultant protein profile was analyzed and at least 154 proteins in cholecyst-derived extracellular matrix (CDE) and 186 proteins in jejunum-derived extracellular matrix (JDE) were identified. Both the matrices contained several extracelluar proteins including fibronectin, nidogen, decorin, and lumican that are known to participate in wound healing responses. However, the CDE had fewer cellular proteins than JDE, especially the latter contained class-I and class-II histocompatibility antigens which are incriminated as potent immunogens responsible for graft rejection. The results of the study suggested that the ECMs used for the scaffold preparation need not be "acellular" and differences in the protein composition of the ECMs might have caused the differential wound healing responses. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 489-496, 2017.

Increased sensitivity of BRCA defective triple negative breast tumors to plumbagin through induction of DNA Double Strand Breaks (DSB).

We have earlier shown that Plumbagin (PB) can induce selective cytotoxicity to BRCA1 defective ovarian cancer cells; however, the effect of this molecule in BRCA1 mutated breast cancers has not been analyzed yet. Here, we report that reactive oxygen species (ROS) induced by PB resulted in DNA DSB and activates downstream signaling by ATR/ATM kinases and subsequent apoptosis. PB reduces DNA- dependent protein kinase (DNA-PK) expression and inhibits NHEJ (Non Homologous End Joining) activity in BRCA1 defective breast cancer cells. Also, PB induces apoptosis in two different BRCA1 conditional knock out murine models: MMTV-Cre; BRCA1(Co/Co) and WAP-Cre; BRCA1(Co/Co), at 2 mg/kg body weight, but 32 mg/kg of carboplatin (CN) was needed to induce apoptosis in them. This is the first study where two different tissue specific promoter driven transgenic mice models with BRCA1 exon 11 deletions are used for preclinical drug testing. The apoptosis induced by PB in HR (Homologous Recombination) defective triple negative BRCA1 mutant cell lines and in mouse models occur by inducing ROS mediated DNA DSB. The toxicity profile as compared with CN in transgenic mice provides evidence for PB's safer disposition as a therapeutic lead in breast cancer drug development.

Fibroblast-loaded cholecyst-derived scaffold induces faster healing of full thickness burn wound in rabbit.

Graft-assisted healing is often proposed for clinical management of large-sized third-degree cutaneous burn wounds. Skin-graft substitutes prepared by loading appropriate cell types on suitable scaffolds have been found successful. We have previously shown that cholecyst-derived scaffold prepared by a non-detergent/enzymatic method can be used as skin-graft substitute for promoting healing of full thickness excision wounds in rabbit. This article examines the use of this scaffold for preparing bio-artificial grafts by loading homologous fibroblasts. The healing potential was evaluated in a rabbit model of full thickness skin-burn wound. The healing process was evaluated by gross morphology evaluation and histomorphology evaluation at 7, 14 and 28 days of healing. Ex vivo imaging of the wounded tissue was performed and it was found that the loaded fibroblasts remained viable at least for 14 days in the healing wound. By the first week, re-epithelialisation was evident in all animals treated with the cell-loaded graft. Histomorphological wound healing parameters such as the quickness of re-epithelialisation, the nature of collagen deposition and the extent of neo-vascularisation indicated that cell-loaded grafts promoted faster healing of the wounds. Results of immunohistochemistry indicated a parallel change in the number of proliferating cells and myofibroblast in the healing tissue. Although the pathophysiology of the healing reaction was not established, the observations suggested that homologus fibroblast-loaded cholecyst-derived scaffold promoted faster healing of third-degree wounds in rabbit model by modulating myofibroblast response. It was concluded that cholecyst-derived scaffold prepared by the non-detergent/enzymatic method is a potential scaffold for fabricating bioartificial skin grafts.

Comparative local immunogenic potential of scaffolds prepared from porcine cholecyst, jejunum, and urinary bladder in rat subcutaneous model.

Extracellular matrices isolated from several mammalian organs/tissues have found several clinical uses as xenografts or implants. However, they may cause complications because of adverse immunologic reactions. Scaffolds that promote favorable graft-acceptance reaction are preferred for fabricating xenografts. The objective of this study was to evaluate the immunogenic potential of a porcine cholecyst-derived scaffold (CDS), prepared by a non-detergent/enzymatic method, in comparison with jejunum and urinary bladder-derived scaffolds in a rat subcutaneous model. Key graft-rejection/acceptance reaction was evaluated at the site of implantation by studying the occurrence and/or function of immunocompetent cells in the tissue reaction. There was differential occurrence of M1-macrophage, M2-macrophage, T-helper cells, T-cytotoxic cells, B-cells, and mast cells in the tissue reaction and the CDS attracted few cells compared with other scaffolds. Real-time polymerase chain reaction for evaluating mRNA of functional markers like inducible nitric oxide synthase (M1 macrophage), arginase 1 (M2 macrophage), interferon gamma (TH1 lymphocytes), and interlukin-4 (TH2 lymphocytes) suggested that the CDS, compared with the scaffolds prepared from small intestine and urinary bladder, elicited M2 macrophage and TH2 lymphocyte polarization that are congenial graft-acceptance reactions. The results indicated that CDS has less immunogenic potential compared with the scaffolds prepared from jejunum and urinary bladder when used as subcutaneous graft in rats. It was concluded that CDS is a promising animal-derived xenograft for biomedical application.

Biocompatibility and Immunophenotypic Characterization of a Porcine Cholecyst-derived Scaffold Implanted in Rats.

Comparative histomorphological assessment of local response to implanted reference biomaterial, also called biocompatibility testing/evaluation, in an appropriate animal model is a widely practiced safety evaluation procedure performed on biomaterials before clinical use. Standardized protocols and procedures, originally designed for testing synthetic materials, available for the testing/evaluation do not account for the immunogenic potential of a candidate biomaterial. Therefore, it is appropriate to supplement the routine biocompatibility test reports with adjunct data that may provide insight into the immunogenic potential of candidate biomaterials, especially when testing biomaterials that are derived from mammalian sources. This article presents expanded safety evaluation data of a porcine cholecyst-derived scaffold (CDS) intended as a xenogeneic graft. The biocompatibility was tested in rat subcutaneous model in comparison with a reference material and the CDS was found biocompatible. However, when studied by immunohistochemistry and real-time reverse transcription polymerase chain reaction for the number and/or polarization of M1 macrophage, M2 macrophage, cytotoxic T-cell, helper T cell, TH1 cell, and TH2 cell, the CDS appeared to induce a differential local immunopathological tissue reaction despite the similarity in biocompatibility with the reference material. The adjunct data collected were useful for objectively assessing the safety of CDS as a xenograft.

Wound healing potential of scaffolds prepared from porcine jejunum and urinary bladder by a non-detergent/enzymatic method.

Scaffolds prepared using extracellular matrices of mammalian organs/tissues, when used as grafts, have wound healing potential. This paper evaluated the physical properties and in vivo wound healing potential of jejunum-derived scaffold (JDS) and urinary bladder-derived scaffold (UDS) of porcine origin prepared by a non-detergent/enzymatic method. The former had higher flexural rigidity and suture retention strength compared to the latter, but both of them had the essential flexural rigidity and suture retention strength required for skin grafts. Full thickness skin-wounds on rabbit dorsum were treated with these scaffolds and the wound healing ability was compared by studying histomorphology parameters such as re-epithelialisation, collagen deposition, angiogenesis, proliferation of cells, mesenchymal cell infiltration and myofibroblast response. The extent of these reactions was assessed using histomorphometry. The results indicated that both grafts initiated healing faster than those wounds without any graft, as evidenced by the extent of cell proliferation and mesenchymal cell infiltration. The myofibroblast response persisted longer in the non-graft assisted wound healing reaction compared to the healing in the graft assisted wounds. Moreover, the JDS induced higher cell proliferation and greater angiogenesis than UDS probably indicating better healing by the former. The results suggested that JDS and UDS prepared by non-detergent/enzymatic method have potential clinical applications.

Biomaterial properties of cholecyst-derived scaffold recovered by a non-detergent/enzymatic method.

Isolation procedures for the recovery of extracellular matrices (ECMs) from animal organs/tissues that are useful in regenerative medicine involve multiple sequential steps/stages including collection of the source organ at slaughter, their transportation to laboratory, decellularization, decontamination, stabilization, and sterilization. Most of these steps require extensive use of chemicals/reagents/enzymes which may also adversely affect the quality of the scaffold. With an effort to minimize the use of chemicals/reagents/enzymes, while extracting biomaterial-grade ECM from porcine cholecyst (gall bladder), we performed preisolation ex situ incubation of the organ in a stabilizing agent that also caused in situ crosslinking of tissue-components and delaminated the collagen-rich ECM from the tissue-layer beneath the mucosa. The physical, chemical, and biological properties of the isolated scaffolds were similar to that of a commercially available porcine small intestinal submucosa. The cholecyst-derived scaffold not only satisfied preclinical safety-test procedures such as cytotoxicity, local response, and endotoxin load but also showed the potential to promote healing of full-thickness skin wound in a rabbit model. The procedure was also suitable for isolating scaffolds from other hollow organs such as jejunum and urinary bladder. It was concluded that enzyme/detergent treatment may be an avoidable step while isolating biomaterial-grade scaffolds from hollow organs.