Search results for: collagenase

Authors: Ozlem Bahadir Acikara, Mert Ilhan, Ekin Kurtul, Karel Smejkal, Esra Kupeli Akkol

Abstract:

Present study is aimed to investigate in vitro inhibitory effects of the extracts prepared from the aerial parts of Podospermum canum (Asteraceae) on hyaluronidase, collagenase, and elastase enzymes using a bioassay-guided fractionation. Inhibitory effects of the extract, sub-extracts, fractions obtained by column chromatography, and isolated compounds on collagenase, elastase, and hyaluronidase were performed by using in vitro enzyme inhibitory assays based on spectrophotometric evaluation. The ethyl acetate and remaining water extracts prepared from the plant displayed significant inhibitory activities on collagenase and elastase, while petroleum ether and chloroform extracts did not show any inhibitory activity. Eleven known compounds: arbutin, 6'-O-caffeoylarbutin, cichoriin, 3,5-dicaffeoylquinic acid methyl ester, apigenin-7-O-β-glucoside, luteolin-7-O-β-glucoside, apigenin-7-O-β-rutinoside, isoorientin, orientin, vitexin, procatechuic acid, and compound 4-hydroxy-benzoic acid 4-(6-O-α-rhamnopyranosyl-β-glucopyranosyl) benzyl ester have been obtained from ethyl acetate sub-extract of the plant through bioassay-guided fractionation and isolation. Results of the present study have revealed that among the isolated compounds, apigenin-7-O-β-glucoside, luteolin-7-O-β-glucoside, apigenin-7-O-β-rutinoside and isoorientin showed potent enzyme inhibitory activities. However, methanolic extract of P. canum displayed a greater inhibitory activity than fractions and isolated compounds both on collagenase and elastase.

Keywords: Asteraceae, collagenase, elastase, hyaluronidase, Podospermum canum

Procedia PDF Downloads 96

Authors: Anthony Barker, Roland Jiang

Abstract:

Dupuytren’s contractures are a debilitating condition affecting the palmar fascia of the hand reducing its function. This case series looks at the minimally-invasive technique of Xiaflex injections and the outcome in a public health setting. 15 patients undertook collagenase injection (Xiaflex, C. histolyticum) injection over the period from September 2015 to May 2017 at Fairfield Hospital, NSW. Their reported outcome post injection and in follow-up was recorded as well as their satisfaction and likelihood to request the procedure in the future. Other treatment modalities include percutaneous needle aponeurotomy, limited palmar fasciotomy, and palmar fasciectomy. A literature review of cost-effectiveness was performed to compare Xiaflex suitability for waitlist reduction in a public setting given average waiting times in the public setting extend past 365 days.

Keywords: Dupuytrens Disease, xiaflex, collagenase, plastic surgery

Procedia PDF Downloads 144

Authors: Yong Cang

Abstract:

RJV001 is a subcutaneous injection containing mutated recombinant Collagenase H (ColH), leading to disruption of collagen matrix in adipose tissue and programmed cell death of adipocytes. Here we reported our clinical investigation of the safety, tolerance and pharmacodynamics of localized RJV001 injection into healthy human abdominal fat tissues (NCT04821648, Arizona Research Center). Investigate the safety, tolerance and clinical pharmacodynamics of subcutaneous RJV001 in humans. In the dose-escalating study, 18 subjects completed the study, 100% female, 78% white, with a mean age of 42[±9.9]. All three tested dose (0.05, 0.075 and 0.15 mg/injection), up to 30 injections, were safe and well-tolerated. Bruising and tenderness to palpation, mild to moderate, were the most frequent local skin reactions but nearly all resolved within 30 days. Additionally, physician-monitored ultrasound measurement showed that a reduction in abdominal fat tissue thickness was consistently observed in Cohort C (0.075, 0.15 mg/injection, 30injections), with a mean reduction of 7.37 [± 2.020] mm. Based on this clinical study, RJV001 has been advanced to phase II clinical studies. In the dose-escalating study, subcutaneously administered RJV001 was safe and well-tolerated in healthy adults up to 0.15 mg/injection, 30 injections. Fat reduction and adipocytolysis were observed by ultrasound measurements and histological analysis for exploratory purposes.

Keywords: fat reduction, mutant collagenase, clinical trial, subcutaneous injection

Procedia PDF Downloads 76

Authors: G. Krishnamoorthy, S. Anandhakumar

Abstract:

The effect of D-Lysine (D-Lys) on collagen with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide(EDC)/N-hydroxysuccinimide(NHS) initiated cross linking using experimental and modelling tools are evaluated. The results of the Coll-D-Lys-EDC/NHS scaffold also indicate an increase in the tensile strength (TS), percentage of elongation (% E), denaturation temperature (Td), and decrease the decomposition rate compared to L-Lys-EDC/NHS. Scanning electron microscopic (SEM) and atomic force microscopic (AFM) analyses revealed a well ordered with properly oriented and well-aligned structure of scaffold. The D-Lys stabilizes the scaffold against degradation by collagenase than L-Lys. The cell assay showed more than 98% fibroblast viability (NIH3T3) and improved cell adhesions, protein adsorption after 72h of culture when compared with native scaffold. Cell attachment after 74h was robust, with cytoskeletal analysis showing that the attached cells were aligned along the fibers assuming a spindle-shape appearance, despite, gene expression analyses revealed no apparent alterations in mRNA levels, although cell proliferation was not adversely affected. D-Lysine (D-Lys) plays a pivotal role in the self-assembly and conformation of collagen fibrils. The D-Lys assisted EDC/NHS initiated cross-linking induces the formation of an carboxamide by the activation of the side chain -COOH group, followed by aminolysis of the O-iso acylurea intermediates by the -NH2 groups are directly joined via an isopeptides bond. This leads to the formation of intra- and inter-helical cross links. Modeling studies indicated that D-Lys bind with collagen-like peptide (CLP) through multiple H-bonding and hydrophobic interactions. Orientational changes in collagenase on CLP-D-Lys are observed which may decrease its accessibility to degradation and stabilize CLP against the action of the former. D-Lys has lowest binding energy and improved fibrillar-assembly and staggered alignment without the undesired structural stiffness and aggregations. The proteolytic machinery is not well equipped to deal with Coll-D-Lys than Coll-L-Lys scaffold. The information derived from the present study could help in designing collagenolytically stable heterochiral collagen based scaffold for biomedical applications.

Keywords: collagen, collagenase, collagen like peptide, D-lysine, heterochiral collagen scaffold

Procedia PDF Downloads 365

Authors: Kumari Rekha, Mathur K Dhananjay, Maheshwari Deepanshu, Nautiyal Nidhi, Shubham Smriti, Laal Deepika, Sinha Swati, Kumar Anupam, Biswas Subhrajit, Shiv Kumar Sarin

Abstract:

Background: Mesenchymal stem cells are multipotent stem cells derived from mesoderm and are used for therapeutic purposes because of their self-renewal, homing capacity, Immunomodulatory capability, low immunogenicity and mitochondrial transfer signaling. MSCs have the ability to regulate the mechanism of both innate as well as adaptive immune responses through the modulation of cellular response and the secretion of inflammatory mediators. Different sources of MSC are UC MSC, BM MSC, Dental Pulp, and Adipose MSC. The most frequent source used is umbilical cord tissue due to its being easily available and free of limitations of collection procedures from respective hospitals. The immunosuppressive role of MSCs is particularly interesting for clinical use since it confers resistance to rejection by the host immune response. Methodology: In this study, T helper cells (TH4), Cytotoxic T cells (CD-8), immunoregulatory cells (CD25 +FOXP3+) are compared from isolated MSC from three different methods, UC Dissociation Kit (Miltenyi), Explant Culture and Collagenase Type-IV. To check the immunomodulatory property, these MSCs were seeded with PBMC(Coculture) in CD3 coated 24 well plates. Cd28 antibody was added in coculture for six days. The coculture was analyzed in FACS Verse flow cytometry. Results: From flow cytometry analysis of coculture, it found that All over T helper cells (CD4+) number p<0.0264 increases in (All Enzymes) MSC rather than explant MSC(p>0.0895) as compared to Collagenase(p>0.7889) in a coculture of Activated T cell and Mesenchymal Stem Cell. Similar T reg cells (CD25+, FOXP3+) expression p<0.0234increases in All Enzymes), decreases in Explant and Collagenase. Experiments have shown that MSCs can also directly prevent the cytotoxic activity of CD8 lymphocytes mainly by blocking their proliferation rather than by inhibiting the cytotoxic effect. And promoting the t-reg cells, which helps in the mediation of immune response in various diseases. Conclusion: MSC suppress Cytotoxic CD8 T cell and Enhance immunoregulatory T reg (CD4+, CD25+, FOXP3+) Cell expression. Thus, MSC maintains a proper balance(ratio) between CD4 T cells and Cytotoxic CD8 T cells.

Keywords: MSC, disease, T cell, T regulatory

Procedia PDF Downloads 74

Authors: Soumya S., Manju Nidagodu Jayakumar, Vellore Kannan Gopinath

Abstract:

Dental pulp inflammation resulting from dental caries often leads to a pathologic condition known as irreversible pulpitis and the currently managed by root canal treatment. Extirpation of the entire pulp tissue is done during this procedure, and the canal space is filled with synthetic materials. Recent studies in the stem cell biology state that some portion of the irreversibly inflamed pulp tissue could be viable with progenitor cells, having the properties similar to that of Mesenchymal stem cells. Hence, we aim to isolate Dental Pulp Stem Cells (DPSCs) from patients diagnosed with severe irreversible pulpitis and characterize the cells for the MSC specific markers. The pulp tissue was collected from the dental clinic and subjected to collagenase/dispase digestion. The isolated cells were expanded in culture, and the phenotypic characterization was done using flow cytometry. MSC specific markers such as CD-90, CD-73, and CD-105 were analysed along with negative markers such as CD-14 and CD-45. The isolated cells expressed positive expression for CD markers with CD90 and CD105 ( > 95%) and CD73 (19%). The cells did not express the negative markers CD-14 and CD-45. The commercially available DPSCs from vital extracted teeth, preferably molar/wisdom teeth with large pulp cavity or incomplete root growth in young patients (aged 15-30 years) showed more than 90% expression for all the CD markers such as CD-90, 73 and 105, whereas negative for CD-14 and CD-45. The DPSCs isolated from inflamed pulp tissue showed a less expression for CD-73 compared to the commercially available DPSCs whereas, as the other two markers were found to show similar percentage of positive expression. This could be attributed to the fact that the pulp population is very heterogeneous and we used the pooled tissue from different patients. Hence the phenotypic characterization and comparison with the commercially available DPSCs proved that the inflamed pulp tissue is a good source of MSC like cells which can be utilized further for regenerative application.

Keywords: collagenase/dispase, dental pulp stem cells, flow cytometry, irreversible pulpitis

Procedia PDF Downloads 216

Authors: M. Khallaf, R. Khalil, H. Ghetas

Abstract:

In the present study, V. harveyi and V. alginolyticus were isolated from cultured seabass and seabream at Damietta Governorate, Egypt, during summer season. Isolates were biochemically and molecularly identified using primers for Vhh and Collagenase genes. The most prominent clinical observations of diseased fish were exophthalmia, abdominal distension, and multifocal cutaneous hemorrhagic ulceration on the dorsal musculature and caudal peduncle. Physicochemical characteristics of water samples indicated that the unionized ammonia, nitrate, and hydrogen sulphate concentrations were higher than the acceptable limits. Heavy metals concentrations in water samples exhibited higher concentrations than the permissible levels for fish culture, which was considered as chemical stressors that increase the prevalence of these bacterial diseases. Immune parameters were lower in diseased seabass and seabream than apparently healthy fish. Lesions of different fish organs were identified histopathologically.

Keywords: seabass, seabream, Vibrio alginolyticus, Vibrio harveyi

Procedia PDF Downloads 90

Authors: Rauza Sukma Rita, Katsuya Dezaki, Yuko Maejima, Toshihiko Yada

Abstract:

Oxytocin is a nine-amino acid peptide synthesized in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the hypothalamus. Oxytocin promotes contraction of the uterus during birth and milk ejection during breast feeding. Although oxytocin receptors are found predominantly in the breasts and uterus of females, many tissues and organs express oxytocin receptors, including the pituitary, heart, kidney, thymus, vascular endothelium, adipocytes, osteoblasts, adrenal gland, pancreatic islets, and many cell lines. On the other hand, in pancreatic islets, oxytocin receptors are expressed in both α-cells and β-cells with stronger expression in α- cells. However, to our knowledge there are no reports yet about the effect of oxytocin on cytosolic calcium reaction on α and β-cell. This study aims to investigate the effect of oxytocin on α-cells and β-cells and its oscillation pattern. Islet of Langerhans from wild type mice were isolated by collagenase digestion. Isolated and dissociated single cells either α-cells or β-cells on coverslips were mounted in an open chamber and superfused in HKRB. Cytosolic concentration ([Ca2+]i) in single cells were measured by fura-2 microfluorimetry. After measurement of [Ca2+]i, α-cells were identified by subsequent immunocytochemical staining using an anti-glucagon antiserum. In β-cells, the [Ca2+]i increase in response to oxytocin was observed only under 8.3 mM glucose condition, whereas in α-cells, [Ca2+]i an increase induced by oxytocin was observed in both 2.8 mM and 8.3 mM glucose. The oscillation incidence was induced more frequently in β-cells compared to α-cells. In conclusion, the present study demonstrated that oxytocin directly interacts with both α-cells and β-cells and induces increase of [Ca2+]i and its specific patterns.

Keywords: α-cells, β-cells, cytosolic calcium concentration, oscillation, oxytocin

Procedia PDF Downloads 156

Authors: Lavrentiadou S. N., Angelou V., Chatzimisios K., Papazoglou L.

Abstract:

The aim of this study was the isolation and culture of keratinocytes and fibroblasts from feline skin to ultimately create an artificial engineered skin (including dermis and epidermis) useful for the effective treatment of large cutaneous deficits in cats. Epidermal keratinocytes and dermal fibroblasts were freshly isolated from skin biopsies using an 8 mm biopsy punch obtained from 8 healthy cats that had undergone ovariohysterectomy. The owner’s consent was obtained. All cats had a complete blood count and a serum biochemical analysis and were screened for feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) preoperatively. The samples were cut into small pieces and incubated with collagenase (2 mg/ml) for 5-6 hours. Following digestion, cutaneous cells were filtered through a 100 μm cell strainer, washed with DMEM, and grown in DMEM supplemented with 10% FBS. The undigested epidermis was washed with DMEM and incubated with 0.05% Trypsin/0.02% EDTA (TE) solution. Keratinocytes recovered in the TE solution were filtered through a 100 μm and a 40 μm cell strainer and, following washing, were grown on a collagen type I matrix in DMEM: F12 (3:1) medium supplemented with 10% FΒS, 1 μm hydrocortisone, 1 μm isoproterenol and 0.1 μm insulin. Both fibroblasts and keratinocytes were grown in a humidified atmosphere with 5% CO2 at 37oC. The medium was changed twice a week and cells were cultured up to passage 4. Cells were grown to 70-85% confluency, at which point they were trypsinized and subcultured in a 1:4 dilution. The majority of the cells in each passage were transferred to a freezing medium and stored at -80oC. Fibroblasts were frozen in DMEM supplemented with 30% FBS and 10% DMSO, whereas keratinocytes were frozen in a complete keratinocyte growth medium supplemented with 10% DMSO. Both cell types were thawed and successfully grown as described above. Therefore, we can create a bank of fibroblasts and keratinocytes, from which we can recover cells for further culture and use for the generation of skin equivalent in vitro. In conclusion, cutaneous cell isolation and cell culture and expansion were successfully developed. To the authors’ best knowledge, this is the first study reporting isolation and culture of keratinocytes and fibroblasts from feline skin. However, these are preliminary results and thus, the development of autologous-engineered feline skin is still in process.

Keywords: cat, fibroblasts, keratinocytes, skin equivalent, wound

Procedia PDF Downloads 77

Authors: K. J. Keerthi, Vasundhara Kamineni, A. Ravi Shanker, T. Rammurthy, A. Vijaya Lakshmi, Q. Hasan

Abstract:

Pancreatic β-cells are the predominant insulin-producing cell types within the Islets of Langerhans and insulin is the primary hormone which regulates carbohydrate and fat metabolism. Apoptosis of β-cells or insufficient insulin production leads to Diabetes Mellitus (DM). Current therapy for diabetes includes either medical management or insulin replacement and regular monitoring. Replacement of β- cells is an attractive treatment option for both Type-1 and Type-2 DM in view of the recent paper which indicates that β-cells apoptosis is the common underlying cause for both the Types of DM. With the development of Edmonton protocol, pancreatic β-cells allo-transplantation became possible, but this is still not considered as standard of care due to subsequent requirement of lifelong immunosuppression and the scarcity of suitable healthy organs to retrieve pancreatic β-cell. Fetal pancreatic cells from abortuses were developed as a possible therapeutic option for Diabetes, however, this posed several ethical issues. Hence, in the present study Mesenchymal stem cells (MSCs) were differentiated into insulin producing cells which were isolated from Human Umbilical cord (HUC) tissue. MSCs have already made their mark in the growing field of regenerative medicine, and their therapeutic worth has already been validated for a number of conditions. HUC samples were collected with prior informed consent as approved by the Institutional ethical committee. HUC (n=26) were processed using a combination of both mechanical and enzymatic (collagenase-II, 100 U/ml, Gibco ) methods to obtain MSCs which were cultured in-vitro in L-DMEM (Low glucose Dulbecco's Modified Eagle's Medium, Sigma, 4.5 mM glucose/L), 10% FBS in 5% CO2 incubator at 37°C. After reaching 80-90% confluency, MSCs were characterized with Flowcytometry and Immunocytochemistry for specific cell surface antigens. Cells expressed CD90+, CD73+, CD105+, CD34-, CD45-, HLA-DR-/Low and Vimentin+. These cells were differentiated to β-cells by using H-DMEM (High glucose Dulbecco's Modified Eagle's Medium,25 mM glucose/L, Gibco), β-Mercaptoethanol (0.1mM, Hi-Media), basic Fibroblast growth factor (10 µg /L,Gibco), and Nicotinamide (10 mmol/L, Hi-Media). Pancreatic β-cells were confirmed by positive Dithizone staining and were found to be functionally active as they released 8 IU/ml insulin on glucose stimulation. Isolating MSCs from usually discarded, abundantly available HUC tissue, expanding and differentiating to β-cells may be the most feasible cell therapy option for the millions of people suffering from DM globally.

Keywords: diabetes mellitus, human umbilical cord, mesenchymal stem cells, differentiation

Procedia PDF Downloads 231

Authors: Elena I. Oprita, Oana Craciunescu, Rodica Tatia, Teodora Ciucan, Reka Barabas, Orsolya Raduly, Anca Oancea

Abstract:

Healing of osteochondral defects requires repair of the damaged articular cartilage, the underlying subchondral bone and the interface between these tissues (the functional calcified layer). For this purpose, developing a single monophasic scaffold that can regenerate two specific lineages (cartilage and bone) becomes a challenge. The aim of this work was to develop variants of biodegradable cross-linked composite hydrogel based on natural polypeptides (gelatin), polysaccharides components (chondroitin-4-sulphate and hyaluronic acid), in a ratio of 2:0.08:0.02 (w/w/w) and mixed with Si-hydroxyapatite (Si-Hap), in two ratios of 1:1 and 2:1 (w/w). Si-Hap was synthesized and characterized as a better alternative to conventional Hap. Subsequently, both composite hydrogel variants were cross-linked with (N, N-(3-dimethylaminopropyl)-N-ethyl carbodiimide (EDC) and enriched with a small bioactive molecule (icariin). The small molecule icariin (Ica) (C33H40O15) is the main active constituent (flavonoid) of Herba epimedium used in traditional Chinese medicine to cure bone- and cartilage-related disorders. Ica enhances osteogenic and chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), facilitates matrix calcification and increases the specific extracellular matrix (ECM) components synthesis by chondrocytes. Afterward, the composite hydrogels were characterized for their physicochemical properties in terms of the enzymatic biodegradation in the presence of type I collagenase and trypsin, the swelling capacity and the degree of crosslinking (TNBS assay). The cumulative release of Ica and real-time concentration were quantified at predetermined periods of time, according to the standard curve of standard Ica, after hydrogels incubation in saline buffer at physiological parameters. The obtained cross-linked composite hydrogels enriched with small-molecule Ica were also characterized for morphology by scanning electron microscopy (SEM). Their cytocompatibility was evaluated according to EN ISO 10993-5:2009 standard for medical device testing. Thus, analyses regarding cell viability (Live/Dead assay), cell proliferation (Neutral Red assay) and cell adhesion to composite hydrogels (SEM) were performed using NCTC clone L929 cell line. The final results showed that both cross-linked composite hydrogel variants enriched with Ica presented optimal physicochemical, structural and biological properties to be used as a natural scaffold able to repair osteochondral defects. The data did not reveal any toxicity of composite hydrogels in NCTC stabilized cell lines within the tested range of concentrations. Moreover, cells were capable of spreading and proliferating on both composite hydrogel surfaces. In conclusion, the designed biodegradable cross-linked composites enriched with Si and Ica are recommended for further testing as natural temporary scaffolds, which can allow cell migration and synthesis of new extracellular matrix within osteochondral defects.

Keywords: composites, gelatin, osteochondral defect, small molecule

Procedia PDF Downloads 148

Authors: Inas Raafat, Azza El Bassiouny, Waldemar L. Olszewsky, Nagui E. Mikhail, Mona Nossier, Nora E. I. El-Bassiouni, Mona Zoheiry, Houda Abou Taleb, Noha Abd El-Aal, Ali Baioumy, Shimaa Attia

Abstract:

Background: Orthotopic liver transplantation is an established treatment for patients with severe acute and end-stage chronic liver disease. The shortage of donor organs continues to be the rate-limiting factor for liver transplantation throughout the world. Hepatocyte transplantation is a promising treatment for several liver diseases and can, also, be used as a "bridge" to liver transplantation in cases of liver failure. Aim of the work: This study was designed to develop a highly efficient protocol for isolation and transplantation of hepatocytes in experimental Lewis rat model to provide satisfactory guidelines for future application on humans.Materials and Methods: Hepatocytes were isolated from the liver by double perfusion technique and bone marrow cells were isolated by centrifugation of shafts of tibia and femur of donor Lewis rats. Recipient rats were subjected to sub-lethal dose of irradiation 2 days before transplantation. In a laparotomy operation the spleen was injected by freshly isolated hepatocytes and bone marrow cells were injected intravenously. The animals were sacrificed 45 day latter and splenic sections were prepared and stained with H & E, PAS AFP and Prox1. Results: The data obtained from this study showed that the double perfusion technique is successful in separation of hepatocytes regarding cell number and viability. Also the method used for bone marrow cells separation gave excellent results regarding cell number and viability. Intrasplenic engraftment of hepatocytes and live tissue formation within the splenic tissue were found in 70% of cases. Hematoxylin and eosin stained splenic sections from 7 rats showed sheets and clusters of cells among the splenic tissues. Periodic Acid Schiff stained splenic sections from 7 rats showed clusters of hepatocytes with intensely stained pink cytoplasmic granules denoting the presence of glycogen. Splenic sections from 7 rats stained with anti-α-fetoprotein antibody showed brownish cytoplasmic staining of the hepatocytes denoting positive expression of AFP. Splenic sections from 7 rats stained with anti-Prox1 showed brownish nuclear staining of the hepatocytes denoting positive expression of Prox1 gene on these cells. Also, positive expression of Prox1 gene was detected on lymphocytes aggregations in the spleens. Conclusions: Isolation of liver cells by double perfusion technique using collagenase buffer is a reliable method that has a very satisfactory yield regarding cell number and viability. The intrasplenic route of transplantation of the freshly isolated liver cells in an immunocompromised model was found to give good results regarding cell engraftment and tissue formation. Further studies are needed to assess function of engrafted hepatocytes by measuring prothrombin time, serum albumin and bilirubin levels.

Keywords: Lewis rats, hepatocytes, BMCs, transplantation, AFP, Prox1

Procedia PDF Downloads 282