Journal articles on the topic 'Xenogenic-free'
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1
Johnston, C., F. Silva, R. Walker, T. Warbington, and A. N. Patel. "Next generation xenogenic-free biologics/reagents." Cytotherapy 16, no. 4 (April 2014): S105. http://dx.doi.org/10.1016/j.jcyt.2014.01.387.
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El-Gamal, Aya. "The effect of activated pure platelete rich plasma (P-PRP) on the proliferation of adipose -derived mesenchymal stem cells (AD-MSCs) as a substitute for fetal bovine serum (FBS)." Mansoura Veterinary Medical Journal 20, no. 1 (March 25, 2019): 35–40. http://dx.doi.org/10.35943/mvmj.2019.01.103.
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The field of stem cells has attracted the attention of many researchers as a hope to treat many incurable diseases because of their ability of self-renewal and differentiation into a specific type of cells that compensates damaged cells. Many studies have been published to confirm their effectiveness but there are some obstacles that limit their clinical applications. One of these obstacles is xenogenic fetal bovine serum (FBS); the main proliferative source for stem cell culture with subsequent risk of infection transmission or immunogenic problems. So, in our study, we aimed to replace the xenogenic FBS with xeno-free blood derivative. We tested the effect of different concentrations of activated pure platelet rich plasma (P-PRP); one of the blood derivatives on proliferation of adipose derived mesenchymal stem cells (AD-MSCs) in comparison to FBS and found that 20% activated P-PRP followed by 10% activated P-PRP increased the proliferation rate of AD-MSCs more than 10% FBS.
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Stanco, Deborah, Christian Caprara, Gianluca Ciardelli, Luca Mariotta, Mauro Gola, Greta Minonzio, and Gianni Soldati. "Tenogenic differentiation protocol in xenogenic-free media enhances tendon-related marker expression in ASCs." PLOS ONE 14, no. 2 (February 12, 2019): e0212192. http://dx.doi.org/10.1371/journal.pone.0212192.
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Brailovskaya, T. V., A. P. Vedyaeva, R. V. Kalinin, E. A. Garibyan, Z. A. Tangieva, and A. M. Deniev. "Augmentation the width of a keratinized attached gingiva in patients with dental implantation." Sechenov Medical Journal, no. 4 (December 30, 2018): 5–15. http://dx.doi.org/10.47093/22187332.2018.4.5-15.
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To date, there has been an increase in the scientific interest in the state of soft tissues surrounding dental implants and their influence on the long-term prognosis of implant treatment. It is known, that the risk factors for the development of periimplantitis include a deficiency or complete absence of an attached keratinized gingiva in the area of implants. The article provides a comparative analysis of various methods of mucogingival surgery in the field of dental implants using free gingival autografts and xenogenic dermal matrices.
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Yamamoto, Kenta, Yoshiki Sato, Kenichi Honjo, Hiroaki Ichioka, Fumishige Oseko, Yoshihiro Sowa, Toshiro Yamamoto, Narisato Kanamura, Tsunao Kishida, and Osam Mazda. "Generation of Directly Converted Human Osteoblasts That Are Free of Exogenous Gene and Xenogenic Protein." Journal of Cellular Biochemistry 117, no. 11 (March 31, 2016): 2538–45. http://dx.doi.org/10.1002/jcb.25546.
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Chang, Yen, Cheng-Kuo Hsu, Hao-Ji Wei, Sung-Ching Chen, Huang-Chien Liang, Po-Hong Lai, and Hsing-Wen Sung. "Cell-free xenogenic vascular grafts fixed with glutaraldehyde or genipin: In vitro and in vivo studies." Journal of Biotechnology 120, no. 2 (November 2005): 207–19. http://dx.doi.org/10.1016/j.jbiotec.2005.06.029.
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Özliseli, Ezgi, Didem Ṣen Karaman, Soumyananda Chakraborti, Anna Slita, Marjaana Parikainen, Cecilia M. Sahlgren, and Jessica M. Rosenholm. "Rational evaluation of human serum albumin coated mesoporous silica nanoparticles for xenogenic-free stem cell therapies." Colloids and Surfaces A: Physicochemical and Engineering Aspects 600 (September 2020): 124945. http://dx.doi.org/10.1016/j.colsurfa.2020.124945.
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Meiring, A., I. Schneider, S. Beasley, and E. Woods. "Scalable Production of Human Mesenchymal Stem Cells in a Novel Bioreactor Using a Xenogenic Free Culture System." Cytotherapy 18, no. 6 (June 2016): S7. http://dx.doi.org/10.1016/j.jcyt.2016.03.017.
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Luhur, Arthur, Daniel Mariyappa, Kristin M. Klueg, Kasun Buddika, Jason M. Tennessen, and Andrew C. Zelhof. "Adapting Drosophila melanogaster Cell Lines to Serum-Free Culture Conditions." G3: Genes|Genomes|Genetics 10, no. 12 (October 7, 2020): 4541–51. http://dx.doi.org/10.1534/g3.120.401769.
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Successful Drosophila cell culture relies on media containing xenogenic components such as fetal bovine serum to support continuous cell proliferation. Here, we report a serum-free culture condition that supports the growth and proliferation of Drosophila S2R+ and Kc167 cell lines. Importantly, the gradual adaptation of S2R+ and Kc167 cells to a media lacking serum was supported by supplementing the media with adult Drosophila soluble extract, commonly known as fly extract. The utility of these adapted cells lines is largely unchanged. The adapted cells exhibited robust proliferative capacity and a transfection efficiency that was comparable to control cells cultured in serum-containing media. Transcriptomic data indicated that the S2R+ cells cultured with fly extract retain their hemocyte-specific transcriptome profile, and there were no global changes in the transcriptional output of cell signaling pathways. Our metabolome studies indicate that there were very limited metabolic changes. In fact, the cells were likely experiencing less oxidative stress when cultured in the serum-free media supplemented with fly extract. Overall, the Drosophila cell culture conditions reported here consequently provide researchers with an alternative and physiologically relevant resource to address cell biological research questions.
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Agrali, Omer Birkan, and Bahar Eren Kuru. "Periodontal treatment in a generalized severe chronic periodontitis patient: A case report with 7-year follow-up." European Journal of Dentistry 09, no. 02 (April 2015): 288–92. http://dx.doi.org/10.4103/1305-7456.156844.
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ABSTRACTThe aim of the periodontal treatment is to provide healthy and functional dentition all through a lifetime. In this report, periodontal treatment of a 42-year-old male patient with generalized severe chronic periodontitis is presented. He received initial periodontal treatment together with adjunctive antimicrobials. The devital teeth were endodontically treated, and free gingival grafts were placed at the inadequate keratinized tissue zones before regenerative surgery. Following the surgical treatment using enamel matrix derivatives and xenogenic bone graft combination, the patient was put on a strict recall program. After 12 months, favorable clinical and radiographical improvements were obtained. The 7-year maintenance of the present case with several initially hopeless teeth has been shown and discussed in this report. It can be concluded that optimum oral hygiene level as well as the positive cooperation of the patient enhanced the success of periodontal treatment results even in extremely severe periodontal destruction.
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Brun, Juliane, Tanja Abruzzese, Bernd Rolauffs, Wilhelm K. Aicher, and Melanie L. Hart. "Choice of xenogenic-free expansion media significantly influences the myogenic differentiation potential of human bone marrow–derived mesenchymal stromal cells." Cytotherapy 18, no. 3 (March 2016): 344–59. http://dx.doi.org/10.1016/j.jcyt.2015.11.019.
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Sachdeva, Shivani, Harish Saluja, Amit Mani, and Pravin Mukhi. "Aesthetic root coverage with acellular dermal matrix allograft: a shield for gingival recession." BMJ Case Reports 14, no. 12 (December 2021): e243895. http://dx.doi.org/10.1136/bcr-2021-243895.
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Awareness about root coverage is increasing in day-to-day clinical practice. Patients are more motivated and concerned about aesthetics nowadays along with the issues of sensitivity of teeth. The conventional flap designs and techniques including lateral sliding flaps, coronally advanced flap, subepithelial connective tissue grafts and free gingival grafts are being adopted for root coverage. The newer material including resorbable and non-resorbable guided tissue regenerative membranes, amniotic membrane, platelet-rich fibrin membrane, enamel matrix derivative protein, xenogenic collagen matrix graft along with the soft tissue substitute like acellular dermal matrix allograft are also being used for recession coverage. The present case report describes a case of 22-year-old female patient with the chief complaint of denudation of gums exposing the root surface over the mandibular left central incisor. The soft tissue substitute acellular dermal matrix allograft was used for root coverage as the patient was not willing to procure an autogenous palatal graft. The results were satisfactory with complete root coverage.
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Lohan, Anke, Benjamin Kohl, Carola Meier, and Gundula Schulze-Tanzil. "Tenogenesis of Decellularized Porcine Achilles Tendon Matrix Reseeded with Human Tenocytes in the Nude Mice Xenograft Model." International Journal of Molecular Sciences 19, no. 7 (July 15, 2018): 2059. http://dx.doi.org/10.3390/ijms19072059.
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Cultivation of autologous human tenocytes in a cell-free xenogenic extracellular tendon matrix (xECM) could present an approach for tendon reconstruction. The aim of this study was to achieve tendon-like tissue formation by implanting decellularized porcine Achilles tendons recellularized with human hamstring tendon-derived tenocytes into nude mice. The structure of decellularized xECM was histologically monitored before being dynamically reseeded with human tenocytes. After 6–12 weeks in vivo, construct quality was monitored using macroscopical and histological scoring systems, vitality assay and quantitative DNA and glycosaminoglycan (GAG) assays. For comparison to tendon xECM, a synthetic polyglycolic acid (PGA) polymer was implanted in a similar manner. Despite decellularized xECM lost some GAGs and structure, it could be recellularized in vitro with human tenocytes, but the cell distribution remained inhomogeneous, with accumulations at the margins of the constructs. In vivo, the xECM constructs revealed in contrast to the PGA no altered size, no inflammation and encapsulation and a more homogeneous cell distribution. xECM reseeded with tenocytes showed superior histological quality than cell-free implanted constructs and contained surviving human cells. Their DNA content after six and 12 weeks in vivo resembled that of native tendon and xECM recellularized in vitro. Results suggest that reseeded decellularized xECM formed a tendon-like tissue in vivo.
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Toledano, Manuel, Manuel Toledano-Osorio, Álvaro Carrasco-Carmona, Cristina Vallecillo, Christopher D. Lynch, María T. Osorio, and Raquel Osorio. "State of the Art on Biomaterials for Soft Tissue Augmentation in the Oral Cavity. Part I: Natural Polymers-Based Biomaterials." Polymers 12, no. 8 (August 18, 2020): 1850. http://dx.doi.org/10.3390/polym12081850.
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Oral soft tissue thickening or grafting procedures are often necessary to cover tooth recession, re-establish an adequate width of keratinized tissue, correct mucogingival deformities improving esthetics, prepare a site for an implant or prosthetics, for ridge preservation procedures, and soft tissue contouring around dental implants. Gingival recession and root or implant exposure are commonly associated and have led to mucogingival deficiencies that have traditionally been treated with free gingival grafts and autogenous soft tissue grafts. The latter represents the gold standard in acquiring a functionally adequate zone of keratinized attached gingiva. However, soft tissue substitutes are more usually employed because they lessen morbidity and abbreviate surgical time. This review is aimed at assessing oral soft tissue augmentation techniques and biomaterials used from existing literature, principally concerning scaffolds from both human and animal-based tissue derivatives matrices. In order to avoid the use of human donor tissue, the xenogenic collagen matrices are proposed for soft tissue augmentation. In general, all of them have provided the remodeling processes and enhanced the formation of new connective tissue within the matrix body.
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Martynova, T. V., and I. N. Alexeyeva. "THE COMPARATIVE CHARACTERISTIC OF FUNCTIONAL ACTIVITY PERITONAL MACROPHAGES AT IMMUNE DAMAGE OF A LIVER OF CELLULAR AND ANTIBODY GENESIS IN MICE." Fiziolohichnyĭ zhurnal 55, no. 1 (February 4, 2009): 36–42. http://dx.doi.org/10.15407/fz55.01.036.
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The aim of present work was to compare the functional activity of peritoneal macrophages (Mf) at T-cellular and antibody induced hepatitis in mice of CBA line. T-cellular hepatitis was caused by concanavalin A (ConA), antibody-induced hepatitis was caused by administration of xenogenic anti-liver antibodies: gamma-globulin fractions of antihepatocytotoxic serum (g-AHCS). It was found that single injection of ConA or g-AHCS caused damage of liver with cytolytic syndrome through 20 hours. Functional activity of Mf in these conditions was significantly different. Application of ConA resulted in the decrease in phagocytosis of latex particles and oxygen-dependent metabolism; application of g-AHCS - to increase of these processes. Weakening of Mf activity may be one of the reasons for the decrease of dead cell eliminations that results in the maintenance of inflammatory reaction. At the same time significant amplification of phagocytic Mf activity may be one of the pathways of free radical endogenic sources increase that causes cell alteration and plays its role as mediators at inflammation.
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De Annuntiis, Carlo, Luca Testarelli, and Renzo Guarnieri. "Use of Xenogenic Collagen Matrices in Peri-Implant Soft Tissue Volume Augmentation: A Critical Review on the Current Evidence and New Technique Presentation." Materials 15, no. 11 (May 31, 2022): 3937. http://dx.doi.org/10.3390/ma15113937.
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Plastic peri-implant surgical procedures aiming to increase soft tissue volume around dental implants have long been well-described. These are represented by: pedicle soft tissue grafts (rotational flap procedures and advanced flap procedures) and free soft tissue grafts (epithelialized, also called free gingival graft (FGG), and non-epithelialized, also called, connective tissue graft (CTG) or a combination of both. To bypass the drawback connected with autologous grafts harvesting, xenogenic collagen matrices (XCM)s and collagen-based matrices derived from porcine dermis (PDXCM)s have been introduced, as an alternative, in plastic peri-implant procedures. Aim: This review is aimed to evaluate and to critically analyze the available evidence on the effectiveness of XCMs and PDXCMs in soft tissue volume augmentation around dental implants. Moreover, a clinical case with a new soft tissue grafting procedure technique (Guided Soft Tissue Regeneration, GSTR) is presented. Material and Methods: An electronic search was performed on the MEDLINE database, SCOPUS, Cochrane Library and Web of Science. The electronic search provided a total of 133 articles. One hundred and twenty-eight not meeting the inclusion criteria were excluded. Seven articles of human randomized clinical trials were selected. A total number of 108 patients were treated with CTG, and 110 patients with XCM. Results: in peri-implant soft tissue augmentation procedures, XCMs seem an effective alternative to CTGs, associated with lower patient morbidity and lower operative times.
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Zambuzzi, Willian Fernando, Rodrigo Cardoso de Oliveira, Felipe Ladeira Pereira, Tânia Mary Cestari, Rumio Taga, and José Mauro Granjeiro. "Rat subcutaneous tissue response to macrogranular porous anorganic bovine bone graft." Brazilian Dental Journal 17, no. 4 (2006): 274–78. http://dx.doi.org/10.1590/s0103-64402006000400002.
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The ideal bone graft must present biocompatibility, osteoconductive and osteoinductive properties, resistance and plasticity. Xenogenic grafts of bovine cancellous bone origin are particularly interesting due to their biologically designed porous structure that enhance both cellular and vascular invasion. The purpose of this study was to evaluate the tissue response induced by bovine macrogranular porous anorganic bone implanted in rat subcutaneous tissue. Forty rats were assigned to 2 groups, as follows: the control group received empty collagen capsules and the test group received subcutaneous implants of the test material. Samples were collected after 10, 20, 30 and 60 days and processed histologically. Histological analysis showed at 10 days a granulomatous inflammatory infiltrate, rich in multinucleated giant cells and free of lymphocytes or plasma cells, similarly to mineralized allograft implanted in rat subcutaneous. In later periods, there was a significant decrease in the inflammatory infiltrate and an increase in fibrosis around graft particles. In conclusion, the test material induced a foreign body-type granuloma with subsequent fibrosis around the graft particles implanted in rat subcutaneous and did not elicit any immune response, thus being considered biocompatible.
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Konoplya, A. I., E. S. Litvinova, N. A. Bystrova, M. S. Razumova, and T. V. Chuyeva. "IMMUNE AND METABOLIC DISTURBANCES IN EXPERIMENTAL ACUTE TOXIC HEPATITIS: CORRECTION BY XENOGENIC AND ALLOGENIC HEPATOCYTES." Russian Journal of Transplantology and Artificial Organs 18, no. 2 (June 25, 2016): 91–98. http://dx.doi.org/10.15825/1995-1191-2016-2-91-98.
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For correspondence: Konoplya Alexander Ivanovich. Address: 3,K. Marx St.,Kursk, 305041,Russian Federation. Tel.: job. (4712) 58-81-76; mob. (910) 317-87-88. E-mail: konoplya51@mail.ru.Aim. To study the corrective effects of allogeneic and xenogeneic hepatocytes on metabolic disturbances in acute liver toxicity.Material and methods. Investigations were carried out on 75 adult male Wistar rats weighing 120–160 g, 15 rats and 25 mice on the 5–6th days after birth. Acute toxic hepatitis (ATH) was modeled by intramuscular injection of carbon tetrachloride at a dose of 3 ml / kg as a 50% solution in olive oil, five times at 24-hour intervals. Isolating xenogeneic (mouse) and allogeneic hepatocytes was performed by method of Berry M.N., Friend D.S. The cell suspension was prepared daily and administered at a concentration of 2 × 106 /kg in recipients with ATH intraperitoneally, five times at 24-hour intervals, simultaneously with the first injection of hepatotropic poison.Results. Intoxication by carbon tetrachloride causes development of the biochemical syndromes of liver damage, activation of the functional metabolic activity of peripheral blood neutrophils and free-radical oxidation, breaks intraerythrocytic metabolism. The introduction of allogeneic hepatocytes in recipients with toxic hepatopathy is more efficiently compared with xenogeneic hepatocytes, it corrects local and systemic metabolic disturbances arising due to the impact of hepatotropic poison. Conclusion. Transplantation of xenogenic hepatocytes, and, to a greater extent, of allogenic hepatocytes in ATH conditions is an effective means to restore the functional metabolic activity of hepatocytes, neutrophils and erythrocytes.
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Lassailly, Francois, Emmanuel Griessinger, and Dominique Bonnet. "“Microenvironmental contaminations” induced by fluorescent lipophilic dyes used for noninvasive in vitro and in vivo cell tracking." Blood 115, no. 26 (July 1, 2010): 5347–54. http://dx.doi.org/10.1182/blood-2009-05-224030.
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Abstract Determining how normal and leukemic stem cells behave in vivo, in a dynamic and noninvasive way, remains a major challenge. Most optical tracking technologies rely on the use of fluorescent or bioluminescent reporter genes, which need to be stably expressed in the cells of interest. Because gene transfer in primary leukemia samples represents a major risk to impair their capability to engraft in a xenogenic context, we evaluated the possibility to use gene transfer–free labeling technologies. The lipophilic dye 3,3,3′,3′ tetramethylindotricarbocyanine iodide (DiR) was selected among 4 near-infrared (NIR) staining technologies. Unfortunately we report here a massive transfer of the dye occurring toward the neighbor cells both in vivo and in vitro. We further demonstrate that all lipophilic dyes tested in this study (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine perchlorate [DiI], DiD, DiR, and PKH26) can give rise to microenvironmental contamination, including when used in suboptimal concentration, after extensive washing procedures and in the absence of phagocytosis or marked cell death. This was observed from all cell types tested. Eventually, we show that this microenvironmental contamination is mediated by both direct cell-cell contacts and diffusible microparticles. We conclude that tracking of labeled cells using non–genetically encoded markers should always be accompanied by drastic cross validation using multimodality approaches.
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Padmasekar, M., N. Lingwal, B. Samikannu, C. Chen, H. Sauer, and T. Linn. "Exendin-4 Protects Hypoxic Islets From Oxidative Stress and Improves Islet Transplantation Outcome." Endocrinology 154, no. 4 (April 1, 2013): 1424–33. http://dx.doi.org/10.1210/en.2012-1983.
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Abstract Oxidative stress produced during pancreatic islet isolation leads to significant β-cell damage. Homeostatic cytokines secreted subsequently to islet transplantation damage β-cells by generating oxygen free radicals. In this study, exendin-4, a glucagon-like peptide-1 analog improved islet transplantation outcome by increasing the survival of diabetic recipient mice from 58% to 100%. We hypothesized that this beneficial effect was due to the ability of exendin-4 to reduce oxidative stress. Further experiments showed that it significantly reduced the apoptotic rate of cultured β-cells subjected to hypoxia or to IL-1β. Reduction of apoptotic events was confirmed in pancreatic islet grafts of exendin-4–treated mice. Exendin-4 enhanced Akt phosphorylation of β-cells and insulin released from them. It even augmented insulin secretion from islets cultivated at hypoxic conditions. Exposure to hypoxia led to a decrease in the activation of Akt, which was reversed when β-cells were pretreated with exendin-4. Moreover, exendin-4 increased the activity of redox enzymes in a hypoxia-treated β-cell line and reduced reactive oxygen species production in isolated pancreatic islets. Recovery from diabetes in mice transplanted with hypoxic islets was more efficient when they received exendin-4. In conclusion, exendin-4 rescued islets from oxidative stress caused by hypoxia or due to cytokine exposure. It improved the outcome of syngenic and xenogenic islet transplantation.
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YATSENKO, Ivan, Sergey SKUBLOV, Ekaterina LEVASHOVA, Olga GALANKINA, and Sergey BEKESHA. "Composition of spherules and lower mantle minerals, isotopic and geochemical characteristics of zircon from volcaniclastic facies of the Mriya lamproite pipe." Journal of Mining Institute 242 (May 25, 2020): 150. http://dx.doi.org/10.31897/pmi.2020.2.150.
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The article presents the results of studying the rocks of the pyroclastic facies of the Mriya lamproite pipe, located on the Priazovsky block of the Ukrainian shield. In them the rock's mineral composition includes a complex of exotic mineral particles formed under extreme reduction mantle conditions: silicate spherules, particles of native metals and intermetallic alloys, oxygen-free minerals such as diamond, qusongite (WC), and osbornite (TiN). The aim of the research is to establish the genesis of volcaniclastic rocks and to develop ideas of the highly deoxidized mantle mineral association (HRMMA), as well as to conduct an isotopic and geochemical study of zircon. As a result, groups of minerals from different sources are identified in the heavy fraction: HRMMA can be attributed to the juvenile magmatic component of volcaniclastic rocks; a group of minerals and xenoliths that can be interpreted as xenogenic random material associated with mantle nodules destruction (hornblendite, olivinite and dunite xenoliths), intrusive lamproites (tremolite-hornblende) and crystalline basement rocks (zircon, hornblende, epidote, and granitic xenoliths). The studied volcaniclastic rocks can be defined as intrusive pyroclastic facies (tuffisites) formed after the lamproites intrusion. Obviously, the HRMMA components formed under extreme reducing conditions at high temperatures, which are characteristic of the transition core-mantle zone. Thus, we believe that the formation of primary metal-silicate HRMMA melts is associated with the transition zone D".
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Stogov, M. V., O. V. Dyuryagina, T. A. Silant'eva, E. A. Kireeva, I. V. Shipitsyna, and M. A. Stepanov. "Preclinical evaluation of the efficacy and safety of a new osteoplastic material of xenogenic origin containing vancomycin or meropenem." Genij Ortopedii 28, no. 4 (August 2022): 565–73. http://dx.doi.org/10.18019/1028-4427-2022-28-4-565-573.
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Bone xenomatrix is an available material for plasty due to its availability and possible significant modification. The purpose was comparative evaluation of the efficacy and safety of xenogenic bone graft material impregnated with antibiotics, vancomycin or meropenem, in an experiment on a model of long bone defect healing in rabbits. Methods The study was performed on 28 male rabbits aged from 8 months to 1.2 years. All animals were modeled with a cavity defect of the right and left distal femoral metaphysis measuring 4 × 4 × 6 mm. Bone matrix blocks of the same size were implanted into the defect cavity. Animals of group 1 (n = 8, control) were implanted with a free “clean” bone block. Animals of group 2 (n = 10) with a bone block saturated with vancomycin. Animals of group 3 (n = 10) with a bone block impregnated with meropenem. To assess the effectiveness and safety of the material, clinical, radiological, pathomorphological, histological, and laboratory methods were used. Results X-ray signs of substitution of the studied materials in the defect in animals of group 1 were noted by 182 days, in group 2 – by 84 days, in group 3 – about 182 days. In each group, there was a complication, arthrosis of the knee joint (one animal in each group). According to the histological study, it was found that in groups 2 and 3, a complete elimination of xenomaterial in the middle part of the defect and its replacement with trabecular bone was noted by 182 days after implantation. The severity of irritating action of the materials in the animals of groups 2 and 3 did not exceed the control value. The laboratory blood tests in the animals of groups 2 and 3 also did not reveal significant differences with group 1. Conclusion The developed osteoplastic materials based on bovine bone xenomatrix, impregnated with vancomycin or meropenem, have acceptable safety and efficacy characteristics.
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De Carvalho, Bruno, Eric Rompen, Geoffrey Lecloux, Peter Schupbach, Emilie Dory, Jean-François Art, and France Lambert. "Effect of Sintering on In Vivo Biological Performance of Chemically Deproteinized Bovine Hydroxyapatite." Materials 12, no. 23 (November 28, 2019): 3946. http://dx.doi.org/10.3390/ma12233946.
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The influence of the manufacturing process on physicochemical properties and biological performance of xenogenic biomaterials has been extensively studied, but its quantification on bone-to-material contact remains poorly investigated. The aim of this study was to investigate the effect of different heat treatments of an experimental chemically-deproteinized bovine hydroxyapatite in vivo in terms of new bone formation and osteoconductivity. Protein-free hydroxyapatite from bovine origin was produced under sub-critical conditions and then either sintered at 820 °C or 1200 °C. Structural and morphological properties were assessed by scanning electron microscopy (SEM), measurement of surface area and X-ray diffractometry (XRD). The materials were then implanted in standardized alveolar bone defects in minipigs and histomorphometric evaluations were performed using non-decalcified sections. Marked topographical differences were observed by SEM analysis. As the sintering temperature of the experimental material increased, the surface area significantly decreased while crystallite size increased. In vivo samples showed that the highly sintered BHA presented a significantly lower percentage of newly formed bone than the unheated one (p = 0.009). In addition, the percentage of bone-to-material contact (BMC) was significantly lowered in the highly sintered group when compared to the unsintered (p = 0.01) and 820 °C sintered (p = 0.02) groups. Non-sintered or sintered at 820 °C BHA seems to maintain a certain surface roughness allowing better bone regeneration and BMC. On the contrary, sintering of BHA at 1200 °C has an effect on its morphological and structural characteristics and significantly modify its biological performance (osteoconductivity) and crystallinity.
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Nguyen, My Thi Ngoc, and Ha Le Bao Tran. "Effect of Modified Bovine Pericardium on Human Gingival Fibroblasts in vitro." Cells Tissues Organs 206, no. 6 (2018): 296–307. http://dx.doi.org/10.1159/000501807.
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Supportive membranes have recently been applied to treat periodontal disease in order to achieve periodontal tissue regeneration. The crucial role of these membranes is to facilitate the restoration of the structural and functional periodontium. Bovine pericardium (BP) is mainly composed of collagen type I, which was demonstrated to have good mechanical properties and biological regenerative potential. Our research aimed to extend the application of membrane derived from BP to periodontal disease treatment. However, the fabrication method to achieve a xenogenic-free membrane with the mechanical properties required for periodontal treatment is rarely mentioned. Therefore, a procedure for the extraction and modification of BP using sodium dodecyl sulfate (SDS) and glutaraldehyde (GA) was developed. BP was harvested and decellularized using different SDS concentrations (0.05–0.3%). GA was used to further modify the membranes to achieve suitable thickness, mechanical strength, and pore size. A combination protocol of 0.15% SDS treatment for 12 h with continuous agitation combined with 0.1% GA for 6 h for membrane fabricating was applied. The modified BP (mBP) had the targeted characteristics, such as 0.2–0.5 mm thickness, approximately 10 MPa in tensile strength, 30% in strain force, and a pore size <5 µm, which is comparable to commercially available collagen membranes. Findings from this study demonstrated that the established method was effective in preparing BP membrane for periodontal treatment while decreasing the concentration of reagents and processing time. Moreover, our modified membrane was found to have no cytotoxicity but supports the migration, attachment, and proliferation of human gingival fibroblasts in vitro. Taken together, these results confirmed that mBP is suitable for application in periodontal disease treatment and regeneration.
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Martin, Maria J., Alysson Muotri, Fred Gage, and Ajit Varki. "An Immunogenic Non-Human Sialic Acid in Human ES Cells." Blood 104, no. 11 (November 16, 2004): 4182. http://dx.doi.org/10.1182/blood.v104.11.4182.4182.
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Abstract The potential of human embryonic stem cells (HESC) to differentiate into any cell type or tissue makes them excellent candidates for therapy of many diseases. To be safe for transplantation, HESC-derived cells and tissues must be free of xenogenic components that could trigger an immune response in the patient. Almost all currently existing HESC cell lines have been in contact with non-human cells (a mouse embryonic fibroblast feeder layer) and/or animal products (such as fetal calf serum or “serum replacements”). These xenogenic culture conditions not only increase the risk of infection by non-human pathogens but also, as we address here, the possibility for HESC to incorporate the immunogenic non-human sialic acid N-glycolylneuraminic acid (Neu5Gc). We used flow cytometry to detect Neu5Gc on surfaces of HESC growing under such standard culture conditions and confirmed its presence by chemical analysis, showing that it represented up to 10% of the total sialic acids in HESC. In embryoid bodies (EB), the first step in HESC differentiation, the percentage of Neu5Gc ranged from 5% to 17%, despite being grown in the absence of the feeder layer and with a reduced concentration of serum. While the HESC could incorporate some Neu5Gc from the feeder layer, the major source appears to be the serum-replacement medium, which was found to be very rich in Neu5Gc (129 nmoles/mL). As many healthy humans have “natural” circulating anti-Neu5Gc antibodies (Tangvoranuntakul P et al. Proc Natl Acad Sci USA2003; 100, 12045–12050), we asked if these antibodies could recognize the HESC growing under the standard conditions and if this binding caused activation of complement. Human IgG and complement factor C3b were indeed detected on HESC cell surfaces after exposure to human sera with high level of anti-Neu5Gc antibodies. Much less IgG or C3b was detected after exposure to human sera with low level such antibodies. To reduce the Neu5Gc content of HESC and EB, we substituted the serum-replacement medium with heat inactivated type AB pooled human serum batches that were also pretested to be very low in natural anti-Neu5Gc antibodies. HESC were able to maintain an undifferentiated state when cultured in such conditions, without losing the ability to differentiate into EB. After a week in the new medium, the percentage of Neu5Gc dropped to 1.2% in the HESC and to 0.2% in the EB. Moreover, this process markedly reduced the IgG and C3b deposition caused by exposure to human sera with high levels of anti-Neu5Gc antibodies. Thus, the metabolic uptake and incorporation of Neu5Gc by HESC growing under the currently accepted culture conditions could trigger an immune response against any HESC-derived transplant, if the recipient has naturally occurring anti-Neu5Gc antibodies. We demonstrate that growth and maintenance of HESC in human serum is feasible, and markedly reduces this risk. In practice, a short-term switch to serum from the transplant recipient should be sufficient to eliminate the Neu5Gc risk. And added advantage of this approach is that one can screen for any allogeneic cytotoxic antibodies that happen to be in the transplant recipient’s serum.
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Aussel, Clotilde, Elodie Busson, Helene Vantomme, Juliette Peltzer, and Christophe Martinaud. "Quality assessment of a serum and xenofree medium for the expansion of human GMP-grade mesenchymal stromal cells." PeerJ 10 (May 30, 2022): e13391. http://dx.doi.org/10.7717/peerj.13391.
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Background Cell-based therapies are emerging as a viable modality to treat challenging diseases, resulting in an increasing demand for their large-scale, high-quality production. Production facilities face the issue of batch-to-batch consistency while producing a safe and efficient cell-based product. Controlling culture conditions and particularly media composition is a key factor of success in this challenge. Serum and Xeno-Free Media (SXFM) represent an interesting option to achieve this goal. By reducing batch to batch variability, they increase Good Manufacturing Practices (GMP)-compliance and safety regarding xenogenic transmission, as compared to fetal bovine serum (FBS) supplemented-media or human platelet lysate supplemented medium. Methods In this study, the isolation, expansion and characteristics including the anti-inflammatory function of human mesenchymal stromal cells (MSC) are compared after culture in MEMα supplemented with human Concentrate Platelet Lysate (hCPL, reference medium) or in MSC-Brew GMP Medium. The latter is a GMP SXFM manufactured in bags under strictly controlled conditions in volumes suitable for expansion to a clinical scale and does not require neither pre-coating of the cell culture units nor the addition of blood derivatives at the isolation step. Results We showed that MSC derived from human bone-marrow and adipose tissue can be successfully isolated and expanded in this SXFM. Number and size of Colony-Forming Unit fibroblast (CFU-F) is increased compared to cells cultivated in hCPL medium. All cells retained a CD90+, CD73+, CD105+, HLADR−, CD34−, CD45− phenotype. Furthermore, the osteogenic and adipocyte potentials as well as the anti-inflammatory activity were comparable between culture conditions. All cells reached the release criteria established in our production facility to treat inflammatory pathologies. Conclusions The use of MSC-Brew GMP Medium can therefore be considered for clinical bioprocesses as a safe and efficient substitute for hCPL media.
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Horke, Alexander, Igor Tudorache, Günther Laufer, Martin Andreas, Jose L. Pomar, Daniel Pereda, Eduard Quintana, et al. "Early results from a prospective, single-arm European trial on decellularized allografts for aortic valve replacement: the ARISE study and ARISE Registry data." European Journal of Cardio-Thoracic Surgery 58, no. 5 (May 9, 2020): 1045–53. http://dx.doi.org/10.1093/ejcts/ezaa100.
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Abstract OBJECTIVES Decellularized aortic homografts (DAH) may provide an additional aortic valve replacement option for young patients due to their potential to overcome the high early failure rate of conventional allogenic and xenogenic aortic valve prostheses. METHODS A prospective, European Union-funded, single-arm, multicentre, safety study was conducted in 8 centres evaluating non-cryopreserved DAH for aortic valve replacement. RESULTS One hundred and forty-four patients (99 male) were prospectively enrolled between October 2015 and October 2018, mean age 33.6 ± 20.8 years; 45% had undergone previous cardiac operations. Mean implanted DAH diameter 22.6 ± 2.4 mm and mean durations for the operation, cardiopulmonary bypass and cross-clamp were 341 ± 140, 174 ± 80 and 126 ± 43 min, respectively. There were 2 early deaths (1 LCA thrombus on day 3 and 1 ventricular arrhythmia 5 h postop) and 1 late death due to endocarditis 4 months postoperatively, resulting in a total mortality of 2.08%. One pacemaker implantation was necessary and 1 DAH was successfully repaired after 6 weeks for early regurgitation following subcoronary implantation. All other DAH were implanted as a free-standing root. After a mean follow-up of 1.54 ± 0.81 years, the primary efficacy end points peak gradient (mean 11.8 ± 7.5 mmHg) and regurgitation (mean 0.42 ± 0.49, grade 0–3) were excellent. At 2.5 years, freedom from explantation/endocarditis/bleeding/stroke was 98.4 ± 1.1%/99.4 ± 0.6%/99.1 ± 0.9%/99.2 ± 0.8%, respectively, with results almost identical to those in an age-matched Ross operation cohort of 212 patients (mean age 34 years) despite DAH patients having undergone >2× more previous procedures. CONCLUSIONS The initial results of the prospective multicentre ARISE trial show DAH to be safe for aortic valve replacement with excellent haemodynamics in the short follow-up period.
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Bushueva, Tatiana V., Ilzira A. Minigalieva, Svetlana V. Klinova, Daria R. Shaikhova, Ivan A. Bereza, Anna M. Amromina, Tatiana V. Mazhaeva, Tatiana N. Shtin, and Julia S. Chernova. "Immunochemical, cytogenetic changes and genetic polymorphism in children living under the exposure to unfavourable environmental factors." Hygiene and sanitation 101, no. 12 (January 12, 2023): 1555–61. http://dx.doi.org/10.47470/0016-9900-2022-101-12-1555-1561.
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Introduction. Studies of biomarkers of effects of susceptibility and sensitivity of the human body to exposure to environmental toxicants are a priority in the development of the hygiene science, individual predisposition to adverse effects of chemicals on DNA and the immune system being of special importance. The objective of our study was to assess immunochemical, cytogenetic changes and genetic polymorphism in children exposed to environmental hazards. Materials and methods. We examined immunochemical marker and functional metabolic changes in selected cells, indicators of the adaptive status of the body and xenogenic poisoning of children environmentally exposed to heavy metals. Gene polymorphism was determined by two detoxification genes (GSTP1 and SOD2). Poisoning was assessed by blood levels of heavy metals. Results. We revealed the presence of autoantibodies to the liver and nervous system in 100% of children. A low level of secretory IgA was observed in 27% of children. The salivary lysozyme level indicates a decrease in the protective function of local immunity by 37.9%. We established a direct relationship between the blood levels of heavy metals and genetic instability in somatic cells of the buccal epithelium. The Ile105Val polymorphism of the GSTP1 gene and the Ala16Val polymorphism of the SOD2 gene were found in 45.9% and 28.4% of children, respectively. Limitations. The paper presents the results of a survey of 3 to 6 years children with allergies attending one preschool. The absence of a control cohort prevents us from comparing our findings with those that could have been obtained for children without allergies and/or living in pollution-free areas. Conclusion. The immunochemical and cytogenetic changes, as well as the genetic polymorphism observed in children are most likely associated with adverse health effects of environmental hazards.
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Kraus, Armin, Manfred Infanger, and Frank Meyer. "What does a (general and abdominal) surgeon need to know on plastic surgery?" Polish Journal of Surgery 91, no. 3 (June 6, 2019): 1–5. http://dx.doi.org/10.5604/01.3001.0013.2365.
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Background: Plastic surgery was first introduced as a sub-specialty of general surgery in Germany in 1978. Since then, this surgical subspecialty/discipline has evolved enormous potential, e.g., in the collaboration with other disciplines such as general and abdominal surgery. Aim: To highlight and summarize the basic potential, technical options and novel aspects of plastic surgery, which are relevant for the common interdisciplinary surgical strategies of plastic and general as well as abdominal surgery in clinical practice. Method: Short and compact narrative review based on i) a selection of relevant references from the medical scientific literature and ii) surgical experiences obtained in daily work. Results (selected corner points): 1) Biological protection procedures in vascular surgery by flap coverage after meticulous debridement with or without autogenic vascular reconstruction are used to overcome infection of a vascular prosthesis, a serious problem, associated with the risk of anastomotic rupture and bleeding by transfer of immunological competence due to tissue coverage and finally to induce healing in the area of an infected vascular prosthesis. 2) Fistula treatment for aorto-tracheal or aorto-duodenal fistulas, a big challenge for the referring general surgeon, can be treated by flap coverage, such as by using the interposition of a pectoralis-major flap and the omentum-majus flap, respectively. 3) With regard to nerve surgery, encouraging results after early microsurgical recurrent laryngeal nerve repair with improved subjective voice quality or concerning reconstitution of respiratory capacity in diaphragmatic palsy has been reported. 4) Lymphatic surgery for lymphedema occurring either primarily due to an absence or lack of lymphatic vessels or secondarily due to infection, trauma, radiation therapy or surgery can be indicated in specialized microsurgical centers, e.g., for surgical repair of the lymphatic pathway by i) the interrupted lymphatic system can be reconstructed by an interposition, or ii) the lymphatic fluid can be drained extraanatomically (e.g., by a lymphatico-venous anastomosis). Further techniques are free lymph-node transplantation included in a free vascularized groin flap or autologous lymphatic-vessel transfer or vein-graft interposition (used for lymphatic-vessel interposition). 5) Mass reduction such as dermolipectomy with subsequent split-thickness is a valuable option, which provides excellent volume reduction. 6) Defect coverage: A. Split- or full-thickness skin grafts are a common method of defect coverage in case that the wound bed is clean and well-vascularized and if there is a lack of donor skin, or if the graft bed is of questionable quality using various allogenic or xenogenic skin substitute materials. B. Further methods offer a wide-range armamentarium of local and free fasciocutaneus and musculocutaneous flaps, e.g., after abdomino-perineal rectum extirpation using the vertical rectus-abdominis myocutaneous flap (VRAM) or propeller flaps according to the “angiosome”. 7) Abdominal wall hernia closure with instable skin coverage, flap closure, either alone or in combination with mesh is superior to mesh closure only. 8) Free flaps: If there is no option for a local or pedicled flap availabe, free flaps can be well used for abdominal wall-defect closure (complication rate in experienced hands, low). Conclusion: Plastic surgery is an indispensable partner for specific surgical problems and clinical situations of general and abdominal surgery, which indicates that each general and abdominal surgeon should be well notified on the great options and surgical techniques, which modern plastic surgery can provide to achieve best outcome and quality of life for the patients by combined expertise of these two surgical disciplines.
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Ikeda, M., S. Yamaguchi, M. Murakami, S. Takaoka, Y. Sakaguchi, S. Yasui, K. Iijima, K. Nanya, H. Onodera, and T. Amano. "OP0008 A NOVEL SITE-SPECIFIC PEGYLATED IL-2 WITH POTENT AND TREG-SELECTIVE ACTIVITY IN VIVO." Annals of the Rheumatic Diseases 81, Suppl 1 (May 23, 2022): 5.2–5. http://dx.doi.org/10.1136/annrheumdis-2022-eular.369.
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BackgroundDecreased regulatory T cells (Tregs) and Treg dysfunction are hallmarks of a various autoimmune and inflammatory diseases. While low-dose IL-2 therapy induces Treg expansion in vivo and has clinical benefits in some diseases (e.g., SLE and chronic graft-versus-host disease [GvHD]), there are many concerns about adverse events due to low Treg-selectivity. Furthermore, frequent dosing is needed due to the short half-life.ObjectivesWe discovered a novel site-specific PEGylated IL-2 variant, KKC80, with high Treg selectivity and a long half-life in vivo, which overcomes the issues of low-dose IL-2 therapy.MethodsBased on the co-crystal structure of wild-type IL-2 and its heterotrimeric receptor (PBD ID: 2ERJ), amino acid residues that were to be PEGylation sites were substituted with oAzZLys, an azide-containing lysine derivative. The PEG molecule was site-specifically attached to oAzZLys-incorporated IL-2 by copper-free click chemistry. The binding property to the IL-2 receptors were measured by surface plasmon resonance (SPR). In vitro, Treg selectivity was evaluated by the IL-2-dependent proliferation activity of Tregs and NK cells from human peripheral blood mononuclear cells (PBMCs). In vivo pharmacological activity after the single subcutaneous administration in cynomolgus monkeys was measured by changes in Treg count and Treg activation status in peripheral blood by flow cytometry. Pharmacokinetic parameters were calculated according to serum PEGylated IL-2 concentration. Efficacy in mouse xenogeneic GvHD model using human PBMC-transplanted NOG mice and in monkey DTH model were evaluated.ResultsA novel PEGylated IL-2, KKC80 (human IL-2 desA1/C125S /I129oAzZLys_W-shaped 80 kDa PEG) was discovered by optimizing the PEGylation site and PEG structure based on Treg selectivity and PK. SPR analysis showed that the binding affinity of KKC80 to CD25 was moderately decreased from wild-type IL-2, while binding affinity of KKC80 to IL-2Rβγ was remarkably decreased due to a significant change of the association rate constant. In vitro, wild-type IL-2 activated both Tregs and NK cells in the same concentration range, whereas KKC80 selectively activated Tregs. The Treg selectivity of KKC80 was comparable to another IL-2 mutein, Fc.IL-2 V91K. KKC80, but not Fc.IL-2 V91K, retained its biological activity, even in the presence of a large amount of recombinant soluble CD25, which mimicked the endogenous decoy receptor for IL-2. In monkeys, KKC80 selectively increased peripheral blood Tregs in a dose-dependent manner; the average maximum rate of increase of Treg count in animals treated with 0.01, 0.03, 0.1, 0.3 and 1 mg/kg was 1.5, 3.5, 28, 50 and 154-fold, respectively. In contrast to Tregs, the rates of increase of conventional CD4+ T, CD8+ T and NK cells were low. The Treg increase peaked on day 8 or 11 and lasted for over day 29. KKC80 showed a more sustained upregulation of functional Treg markers (e.g., Foxp3 and CD25) in comparison to Fc.IL-2 V91K. The half-life of KKC80 was calculated as 83.5 to 150 h. At high doses, inflammation-related adverse effects, including increased CRP (≥0.3 mg/kg) and deterioration of general conditions (1 mg/kg) were observed. In the mouse xenogenic GvHD model, KKC80 ameliorated GvHD symptoms and suppressed multiple tissue inflammation markers. Decreased soluble CD25 and IFN-γ were also confirmed, suggesting Treg-mediated anti-inflammatory effect by KKC80 administration were exerted in vivo. In the monkey DTH model, KKC80 suppressed skin inflammation and antibody production.ConclusionAmong next-generation IL-2 variants, KKC80 showed a best-in-class biological profile for Treg activation. A drastic and sustained increase of Tregs with high Treg-selectivity and anti-inflammatory effects were observed in vivo. These data suggest that in comparison to current IL-2 therapy, KKC80 provides superior therapeutic index and efficacy in patients with autoimmune and inflammatory diseases.Figure 1.Disclosure of InterestsMasahiro Ikeda Employee of: Kyowa Kirin Co., Ltd., Shinpei Yamaguchi Employee of: Kyowa Kirin Co., Ltd., Masumi Murakami Employee of: Kyowa Kirin Co., Ltd., Shigeki Takaoka Employee of: Kyowa Kirin Co., Ltd., Yasuko Sakaguchi Employee of: Kyowa Kirin Co., Ltd., Shunki Yasui Employee of: Kyowa Kirin Co., Ltd., Kousuke Iijima Employee of: Kyowa Kirin Co., Ltd., Kenichiro Nanya Employee of: Kyowa Kirin Co., Ltd., Hideyuki Onodera Employee of: Kyowa Kirin Co., Ltd., Toru Amano Employee of: Kyowa Kirin Co., Ltd.
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Fernando, Adrian F., and Joselito F. David. "Combination of Autologous Protein-Rich Fibrin and Bone Graft: An Invaluable Option for Reconstruction of Segmental Mandibular Defects." Philippine Journal of Otolaryngology-Head and Neck Surgery 28, no. 1 (June 18, 2013): 38–42. http://dx.doi.org/10.32412/pjohns.v28i1.509.
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Dear Editor,
Reconstruction of mandibular defects resulting from ablative surgery for benign and malignant tumors remains a reconstructive challenge. For the past decade, the fibular free flap has been the workhorse for large mandibular defects because of its length, versatility, and ability to be harvested with a skin paddle for soft tissue closure. Although its success rate has continuously improved to almost 95%, donor site morbidity remains a matter of concern.1,2 Bone grafts are already widely used in dental surgery but only as fillers for chipped or marginal defects and not for large segmental mandibular defects. We present a new technique of reconstructing segmental mandibular defects using bone grafts combined with autologous platelet-rich fibrin (PRF), a biomaterial derived intra-operatively from the patient that incorporates leukocytes, platelets, growth factors, and a wide range of glycoproteins in a dense fibrin matrix. Moreover, we describe the essential role of PRF in bone healing and regeneration that offers an invaluable reconstructive option that is free of donor site morbidity without sacrificing the main goal of reconstruction in restoring both form and function.
Keywords: Mandibular reconstruction; segmental mandibular defect; bone graft; autologous Platelet-Rich Fibrin (PRF)
MATERIALS AND METHODS
Subject and Indications
A 23-year-old male underwent reconstruction with allogenic bone graft in combination with autologous platelet-rich fibrin (PRF) for a large segmental angle to parasymphyseal mandibular defect. (Figure 1) As in this example, the authors’ technique for segmental mandibular defect reconstruction using bone grafts with PRF was best performed as a second stage procedure following tumor ablation to prevent contamination from oral cavity secretions. As with any elective procedure, a thorough review of the medical history, control of systemic disease and informed consent were necessary. Patients with contraindications for fibular free flaps, such as history of peripheral vascular disease, unfavorable imaging of the lower extremity, venous insufficiency, and anomalous lower extremity vasculature may benefit from this technique.3 Patients who had failed mandibular reconstruction with other methods such as those reconstructed with single alloplastic material, titanium plate, non-vascularized autologous bone graft, or free flaps are likewise candidates for this option.
Procedure
A submandibular incision was made and a sub-platysmal flap was raised to expose the entire length of the mandibular defect. Apart from the preservation of vital structures in the area, it was important not to violate the oral mucosa to prevent contamination of the reconstruction site. A cortico-cancellous allogenic bone graft (Maxgraft®, Botiss Medical AG , Berlin, Germany) was fitted to the mandibular defect and anchored with bicortical screws (2.5; 2.8 mm, lengths 14-20 mm) to a pre-bended 2.5 mm reconstruction titanium plate (Modus Reco 2.5, Medartis, Hochbergerstrasse Basel Switzerland). The remaining gaps between the inlaid grafts were filled with the remaining cancellous bone and biomimetic composite materials. (Figure 2) Although allogenic bone grafts with cortical and cancellous components are recommended for mandibular ramus and condylar reconstruction as these regions are composed of nearly 100% cortical bone, xenogenic (Cerabone®, Botiss Medical AG , Berlin, Germany) or combined alloplastic material- (Maxresorb®, Botiss Medical AG , Berlin, Germany) bone grafts may be used in other regions. (Figure 3)
Meanwhile, venous blood was simultaneously drawn from the patient and placed in a 10 cc glass collecting tube for single centrifuge processing using a PC-O2 centrifuge (PC-O2, Process, Nice, France). The specific centrifuge processes 8 uncoated tubes using a standard protocol specially manufactured for processing PRF using 33° tube angulation at 2700 RPM’s, soft spin for 12 minutes.4 (Figure 4) At the end of the centrifugation process, three distinct fractions of blood components were produced where the intermediate fraction composed of dense PRF clot was used. The other blood components separated by the centrifugation process- serum or platelet-poor plasma (PPP) and red blood cell concentrates, were respectively situated in the superficial and bottom layers of the collecting tube. The PRF clots were then transferred to a PRF processing box (PRF Box®, Process, Nice, France) to prepare standardized membranes and harvest the PRF exudates in a sterile environment.5 (Figure 5) Collagen material of native pericardium (GTR/GBR) membrane (Jason® membrane, Botiss Medical AG, Berlin, Germany) was placed underneath the graft recipient site and the processed PRF membranes were layered over the graft recipient site to stimulate osteoblastic differentiation and neoangiogenesis.6
The entire recipient site was then enveloped with the collagen material, mechanically securing the autologous PRF in contact with the bone grafts. (Figure 6) This established a membrane barrier for guided bone regeneration (GBR) and guided tissue regeneration (GTR) by preventing growth of undesired cells inside the neomandible and allowing osteogenesis and angiogenesis.7 Commercially available collagen biomaterials vary from native collagen membrane to enhanced Ca/P collagen composite materials such as the 3D-stable collagen graft (Mucoderm®, Botiss Medical AG, Berlin, Germany) with larger available sizes for long mandibular defect coverage. The skin was closed in the usual manner and the patient was initially maintained on a liquid diet, progressing to a soft diet over 2-4 weeks. Plain panoramic radiographs after a week confirmed proper alignment of the bone grafts and monthly radiographic series was recommended for the first 6 months after reconstruction.
RESULTS
Monthly panoramic radiographs for the first 6 months after reconstruction showed absence of bone resorption. A 3D reconstruction CT imaging of the mandible was done after 10 months for placement of three osteo-integrated dental implants. (Figure 7) Bone biopsies were also taken in conjunction with placement of dental implants, and sent to the University of Bonn, Germany for histologic evaluation. (Figure 8) The trichrome-stained specimens showed new mineralized tissues consisting of woven bone characterized by high numbers of distributed osteocytes and irregularly arranged fiber bundles within the new bone matrix confirming bone regeneration. (Figure 9) The latest panoramic radiograph of the patient at 26 months after surgery showed absence of gaps between the bone grafts and their junction with the normal mandible, evincing complete bone regeneration and a successful mandibular reconstruction. (Figure 10) A total of 14 cases of segmental mandibular defects have been reconstructed by the authors using the particular technique from January 2011 to February 2013 with 100% success rate and will be reported as a series in the near future.
DISCUSSION
Advancements in mandibular reconstruction have continued to develop over the past decades. The use of alloplastic materials like titanium plating implants for repairing mandibular defects provided patients with rapid rigid mandibular restoration but was limited by numerous complications such as infection, plate extrusion, and subsequent failure. Recently, the concept of distraction osteogenesis, involving bone distraction with an external mechanical device and progressive lengthening of the bone to allow a gap of new bone during the consolidation phase has also been used for mandibular reconstruction but has been limited by poor scar formation, delayed return to function, and inadequate formation of desired bone length.8 The advent of microvascular reconstructive surgery enabled the transfer of vascularized osseous flaps with the most commonly used fibular free flap showing superior results over non-vascularized bone transfer and better quality of life outcome. However, it did not remain free of donor-site morbidities.9
Tissue engineering led to the development and use of bone grafts that hold promise for the future of head and neck repair.10 Numerous clinical studies demonstrated the utility of tissue engineering in developing bone grafts for mandibular defect reconstruction.11 Such have already been widely used over the past decades in oro-maxillary and dental reconstruction, including the recombinant bone morphogenetic protein (rhBMP-2) that is now used with great success in cleft palate repair, alveolar ridge augmentation, and sinus lift procedures.12 Autogenous bone grafts derived from the patient work through osteogenesis, osteoinduction and osteoconduction. However, such are not recommended because apart from enabling a donor site morbidity-free technique, they are best harvested as microvascular flaps. Allogenic bone grafts on the other hand are cadaveric processed grafts that may be cortical, trabecular, or combined in composition and have both osteoconductive and osteinductive properties. Xenografts or processed animal bone graft are a subgroup of the synthetically manufactured alloplasts known to form new bones from their osteoconductive activities.
The use of autologous PRF is already widely used in combination with bone grafts for dental surgeries but not for large mandibular defects.13 Our reconstructive technique using bone grafts for large segmental mandibular defects emphasizes the important role of PRF with its intrinsic factors and leukocyte contents that release high amounts of growth factors such as TGBß1, PDGF-AB, VEGF and matrix glycoproteins.14 Collagen membrane used to envelop the entire recipient site creates a membrane barrier to prevent the growth of soft tissues and allow angiogenesis within the neomandible.15 Fascia lata may be used as a membrane barrier but defeats the authors’ goal of an absolute donor site morbidity-free procedure. Overall, this particular technique along with gentle tissue handling and avoidance of oral cavity contamination for reconstructing large mandibular defects has been found to enhance bone regeneration capable for osteo-integrated dental implantation.
Generally, the harvesting and processing of autologous PRF is simple and inexpensive. Its use with bone grafts is a good substitute for segmental mandibular reconstruction in patients with contraindications to free flap procedures or in cases where patients simply wish to be free from any donor-site morbidity. However, this technique is limited to defects secondary to trauma and ablation of benign conditions as bone regeneration is expected in approximately 6 to 9 months. Mandibular defects following resection of malignant oral neoplasms are still best reconstructed with fibular free flaps as radiation therapy is warranted at the soonest possible time. Histologic validation of bone regeneration and osteoblastic activity index for the 13 other cases performed by the authors using this particular technique necessitates bone research centers that are capable of advanced bone analysis, and none are locally-available at this time. Meanwhile, this technique of combining autologous PRF in bone grafting remains an innovative and invaluable option for mandibular reconstruction today.
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Тишевская, Н. В., Н. М. Геворкян, and А. А. Позина. "A lyophilized form of xenogeneic total RNA stimulates hematopoiesis in post-radiation myelosuppression." Zhurnal «Patologicheskaia fiziologiia i eksperimental`naia terapiia», no. 4 (December 28, 2021): 42–46. http://dx.doi.org/10.25557/0031-2991.2021.04.42-46.
Full textAbstract:
Введение. Аллогенная суммарная РНК, выделенная из клеток лимфоидных органов, стимулирует регенерацию кроветворной ткани после острого и хронического нарушения кроветворной функции. Цель исследования: 1) доказательство отсутствия ксеногенных ограничений механизмов лимфоцитарного контроля регенеративных процессов на примере гемостимулирующего действия суммарной РНК лимфоцитов бычьей селезенки в отношении кроветворения крыс, подвергшихся гамма-облучению в сублетальной дозе; 2) сравнительный анализ эффективности нативной и лиофилизированной форм указанной РНК. Методика. Работа выполнена на белых нелинейных крысах-самцах массой 200-220 г. Cуммарную РНК выделяли методом фенол - хлороформной экстракции из лимфоидных клеток бычьей селезенки. Для создания исходной миелосупрессии 30 крыс подвергли однократному общему воздействию гамма-излучения с источником 60Co в дозе 6 Гр при мощности дозы 0,1 Гр/с, после чего разделили их на 3 равные группы. Через 2 ч после облучения крысам контрольной группы внутрибрюшинно ввели по 0,5 мл 0,9% NaCl; крысам 2-й группы - нативную суммарную РНК в дозе 30 мкг/100г массы, крысам 3-й группы - лиофилизированную суммарную РНК в аналогичной дозе. На 3-и, 7-е и 12-е сут в периферической крови определяли количество ретикулоцитов, лейкоцитов и тромбоцитов, после чего крысы были выведены из эксперимента с целью исследования костномозгового кроветворения. Через 12 сут в костном мозге определяли количество эритроидных, лимфоидных, мегакариоцитарных и миелоидных клеток. Из костного мозга выделяли эритробластические островки (ЭО) и дифференцировали их на пролиферирующие (ЭО 1,2 классов и реконструирующиеся ЭО) и зрелые (ЭО 3 класса и инволюциирующие ЭО) морфо-функциональные клеточные ассоциации. Результаты. Под влиянием ксеногенной суммарной РНК в периферической крови крыс в 2-3 раза увеличилось количество лейкоцитов и в 1,6-1,75 раза возросло число ретикулоцитов. В костном мозге увеличилось количество пролиферирующих миелоидных и лимфоидных элементов, а также общее число клеток эритроидного ряда. Ксеногенная суммарная РНК стимулировала образование ЭО как на основе контакта свободных костномозговых макрофагов с молодыми эритроидными клетками (ЭО 1 и 2 классов), так и на базе реконструкции (ЭО реконструирующиеся). Сравнительный анализ эффектов нативной и лиофилизированной суммарной РНК не выявил различий между гемопоэтическими показателями у крыс, получивших эти препаратов. Заключение. Суммарная РНК, выделенная из лимфоидных клеток бычьей селезенки, активирует гемопоэз у крыс с постлучевой миелосупрессией, что свидетельствует об отсутствии ксеногенных ограничений у млекопитающих в механизмах лимфоцитарного контроля восстановительных процессов. Лиофилизированная суммарная РНК активирует костномозговое кроветворение в те же сроки и в том же объеме, что и нативная форма. Introduction. Allogeneic total RNA isolated from cells of lymphoid organs stimulates regeneration of hematopoietic tissue after acute and chronic disturbance of hematopoietic function. Aim. 1) To prove the absence of xenogeneic limitation for the lymphocytic regulation of regenerative processes using an example of the hemo-stimulating effect of total RNA from bovine spleen lymphocytes on hematopoiesis in rats exposed to sublethal gamma-irradiation; 2) To perform a comparative analysis of the effectiveness of the native and lyophilized forms of the total RNA. Methods. Experiments were performed on white outbred male rats weighing 200-220 g. Total RNA was isolated from bovine spleen lymphoid cells by phenol-chloroform extraction. To create the initial myelosuppression, 30 rats were exposed to a single general 60Co gamma radiation (6 Gy at 0.1 Gy/s). The rats were then divided into 3 equal groups. Two hrs after irradiation, the rats of the control group were injected intraperitoneally with 0.5 ml of 0.9% NaCl; rats of the second group received native total RNA, 30 μg/100 g body weight, and rats of the third group received lyophilized total RNA at a similar dose. On days 3, 7, and 12, the number of peripheral blood reticulocytes, leukocytes, and platelets was measured. The rats were then sacrificed, and bone marrow hematopoiesis was studied. After 12 days, the number of bone marrow erythroid, lymphoid, megakaryocytic, and myeloid cells was measured. Erythroblastic islets (EIs) were isolated from the bone marrow and differentiated into proliferating (class 1 and 2 EIs and reconstructing EIs) and mature (class 3 EIs and involving EIs) morpho-functional cell associations. Results. Under the influence of xenogeneic total RNA, the number of peripheral blood leukocytes increased by 2-3 times, and the number of reticulocytes increased by 1.6-1.75 times. In the bone marrow, the number of proliferating myeloid and lymphoid cells increased, as did the total number of erythroid cells. Xenogeneic total RNA stimulated formation of EIs, based both on the contact of free bone marrow macrophages with young erythroid cells (class 1 and 2 EIs) and on reconstruction (reconstructing EIs). Comparative analysis of the effects of native and lyophilized total RNA did not reveal differences between hematopoietic parameters in rats that received these agents. Conclusion. Total RNA isolated from bovine spleen lymphoid cells activates hematopoiesis in rats with post-radiation myelosuppression. This indicates the absence of mammalian xenogenic limitation of lymphocytic control of recovery processes. Lyophilized total RNA activates bone marrow hematopoiesis at the same rate and to the same extent as the native form.
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Diuriagina, O. V., M. V. Chepeleva, E. I. Kuznetsova, M. A. Kovinka, and A. N. Nakoskin. "The effect of implantation materials based on equine and bovine xenogenic bone extracellular matrix on the formation of extracellular neutrophilic traps (experimental study)." Genij Ortopedii 26, no. 4 (December 2020): 571–75. http://dx.doi.org/10.18019/1028-4427-2020-26-4-571-575.
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Purpose To study the effect of osteoplastic materials based on the extracellular xenomatrix of bovine and equine bone tissue on the formation of neutrophil extracellular traps (NETs) in the peripheral blood of rabbits in the early post-operative period after implantation. Materials and methods The study was carried out on 18 male rabbits of the Soviet Chinchilla breed, aged from 8 months to 1.2 years, weighing from 3.0 to 4.5 kg. A perforated bone defect of a cylindrical shape measuring 2 x 6 mm in the distal metaphysis of the right and left femurs was modeled in the animals. The rabbits were divided into three groups, six animals each. In group I, the bone defect was left unfilled; in group II, the defect was filled with a bovine bone tissue xenomatrix, and an equine bone tissue xenomatrix was implanted in group III animals. The implantation material had the appearance of a yellowish crumb with a particle size of 0.5– 1 mm. Blood smears stained according to Romanovsky-Giemsa were used for counting extracellular neutrophil traps (NETs). The percentage of neutrophils that passed the stages of nuclear transformation and emitted free chromatin into the extracellular space in the form of network-like structures was calculated. Results On days 3–7 of the experiment, the number of NETs increased in the early stages of NETosis in all groups. There were no significant differences between the groups. In group I, on days 7 and 14, the number of early forms of NETs (stages 1a and 1b) returned to the values of the preoperative period. In groups II and III, normalization of NETs (stage 1a) did not occur, and the content of NETs (stage 1b) returned to the initial level only by day 30 of the experiment. On days 3, 7, 14, the number of mature NETs increased in all groups. The highest values were noted in group II, where the bovine xenogeneic matrix was implanted. Conclusion Implantation materials based on the extracellular matrix of equine and bovine xenogeneic bone stimulate excessive formation of early NETs on days 14–30 of the experimental period in response to xenotransplantation. Xenomaterials of bovine bone tissue, in comparison with xenomaterials of equine bone tissue, induce a more pronounced inflammatory reaction in the nearest time after defect filling, which is manifested by higher production of mature NETs on days 3–14 of the experiment.
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Schmitt, Trevor, Nilabh Kajave, Huan Huan Cai, Linxia Gu, Mohammad Albanna, and Vipuil Kishore. "In vitro characterization of xeno-free clinically relevant human collagen and its applicability in cell-laden 3D bioprinting." Journal of Biomaterials Applications, September 22, 2020, 088532822095916. http://dx.doi.org/10.1177/0885328220959162.
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Collagen type I, commonly derived from xenogenic sources, is extensively used as a biomaterial for tissue engineering applications. However, the use of xenogenic collagen is typically associated with species specific variation in mechanical, structural, and biological properties that are known to influence cellular response and remodeling. In addition, immunological complications and risks of disease transmission are also major concerns. The goal of this study is to characterize a new xeno-free human skin-derived collagen and assess its applicability as a bioink for cell-laden 3 D bioprinting. Four different concentrations of human collagen (i.e., 0.5 mg/mL, 1 mg/mL, 3 mg/mL and 6 mg/mL) were employed for the synthesis of collagen hydrogels. In addition, bovine collagen was used as a xenogenic control. Results from SDS-PAGE analysis showed the presence of α1, α2, and β chains, confirming that the integrity of type I human collagen is maintained post isolation. Polymerization rate and compressive modulus increased significantly with increase in the concentration of human collagen. When comparing two different sources of collagen, the polymerization rate of xenogenic collagen was significantly faster (p < 0.05) than human collagen while the compressive modulus was comparable. Raman spectroscopy showed a large peak in the Amide I band around 1600 cm−1, indicating a dense and supraorganized fibrillar structure in human collagen hydrogels. Conversely, Amide I band intensity for xenogenic collagen was comparable to that of Amide II and Amide III bands. Further, the use of 6 mg/mL human collagen as a bioink yielded 3 D printed constructs with high shape fidelity and cell viability. On the other hand, xenogenic collagen failed to yield stable 3 D printed constructs. Together, the results from this study provides an impetus for using human-derived collagen as a viable alternative to xenogenic sources for 3 D bioprinting of clinically relevant scaffolds for tissue engineering applications.
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Weber, Tilo, Joachim Wiest, Stina Oredsson, and Karen Bieback. "Case Studies Exemplifying the Transition to Animal Component-free Cell Culture." Alternatives to Laboratory Animals, August 19, 2022, 026119292211179. http://dx.doi.org/10.1177/02611929221117999.
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Cell culture techniques are strongly connected with modern scientific laboratories and production facilities. Thus, choosing the most suitable medium for the cells involved is vital, not only directly to optimise cell viability but also indirectly to maximise the reliability of the experiments performed with the cells. Fetal bovine or calf serum (FBS or FCS, respectively) is the most commonly used cell culture medium supplement, providing various nutritional factors and macromolecules essential for cell growth. Yet, the use of FBS encompasses a number of disadvantages. Scientifically, one of the most severe disadvantages is the lot-to-lot variability of animal sera that hampers reproducibility. Therefore, transitioning from the use of these ill-defined, component-variable, inconsistent, xenogenic, ethically questionable and even potentially infectious media supplements, is key to achieving better data reproducibility and thus better science. To demonstrate that the transition to animal component-free cell culture is possible and achievable, we highlight three different scenarios and provide some case studies of each, namely: i) the adaptation of single cell lines to animal component-free culture conditions by the replacement of FBS and trypsin; ii) the adaptation of multicellular models to FBS-free conditions; and (iii) the replacement of FBS with human platelet lysate (hPL) for the generation of primary stem/stromal cell cultures for clinical purposes. By highlighting these examples, we aim to foster and support the global movement towards more consistent science and provide evidence that it is indeed possible to step out of the currently smouldering scientific reproducibility crisis.
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Ngo, Ba Thanh-Truc, Andres Beiras-Fernandez, Claus Hammer, and Eckart Thein. "Hyperacute rejection in the xenogenic transplanted rat liver is triggered by the complement system only in the presence of leukocytes and free radical species." Xenotransplantation, May 2013, n/a. http://dx.doi.org/10.1111/xen.12035.
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Forouzesh, Mehdi, Mojgan Hosseini, Mehran Ataei, Maryam Farzaneh, and Seyed Esmaeil Khoshnam. "An extracellular matrix-based culture system for generation of human pluripotent stem cell derived-hepatocytes." Current Stem Cell Research & Therapy 16 (December 28, 2020). http://dx.doi.org/10.2174/1574888x16666201228144834.
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: Liver disease (hepatic disease) adversely affects the normal function of the liver and causes liver problems. Druginduced liver injury (DILI) can be predicted by primary human hepatocytes. However, the sources of hepatocytes for largescale drug toxicity screening are limited. To solve this problem, pluripotent stem cells (PSCs), mesenchymal stem cells (MSCs), and hepatic stem cells (HSCs) have emerged as attractive cell sources for cell-based therapies. Human PSCs including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have the ability to undergo self-renewal and to differentiate into lineages of ectoderm, mesoderm, and endoderm. Human PSC can be used for generation of hepatocytes to facilitate the development of novel drugs for treatment of severe liver diseases. The therapeutic potential of PSC-derived hepatocytes for liver failure have been identified to enhance the development of chemically defined and xenogenic-free 3D culture methods. To date, several hepatic differentiation strategies and various extracellular matrix (ECM) components have been employed to produce hepatocytes or hepatic-like cells (HLCs) in vitro. In this review, we focused on the potential of Matrigel, collagen type 1, RoGel, and laminin as ECM on the differentiation and function of hESC- and hiPSC-derived hepatocytes. The hepatic differentiation of human ESCs and iPSCs would offer an ideal tool for cell therapy and liver diseases.
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Even, Kayla M., Angela M. Gaesser, Sarah A. Ciamillo, Renata L. Linardi, and Kyla F. Ortved. "Comparing the immunomodulatory properties of equine BM-MSCs culture expanded in autologous platelet lysate, pooled platelet lysate, equine serum and fetal bovine serum supplemented culture media." Frontiers in Veterinary Science 9 (August 25, 2022). http://dx.doi.org/10.3389/fvets.2022.958724.
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Joint injury often leads to cartilage damage and posttraumatic inflammation, which drives continued extracellular matrix degradation culminating in osteoarthritis. Mesenchymal stem cells (MSCs) have been proposed as a biotherapeutic to modulate inflammation within the joint. However, concerns have been raised regarding the immunogenicity of MSCs cultured in traditional fetal bovine serum (FBS) containing media, and the potential of xenogenic antigens to activate the immune system causing rejection and destruction of the MSCs. Xenogen-free alternatives to FBS have been proposed to decrease MSC immunogenicity, including platelet lysate (PL) and equine serum. The objective of this study was to compare the immunomodulatory properties of BM-MSCs culture-expanded in media supplemented with autologous PL (APL), pooled PL (PPL), equine serum (ES) or FBS. We hypothesized that BM-MSCs culture expanded in media with xenogen-free supplements would exhibit superior immunomodulatory properties to those cultured in FBS containing media. Bone marrow-derived MSCs (BM-MSCs) were isolated from six horses and culture expanded in each media type. Blood was collected from each horse to isolate platelet lysate. The immunomodulatory function of the BM-MSCs was assessed via a T cell proliferation assay and through multiplex immunoassay quantification of cytokines, including IL-1β, IL-6, IL-8, IL-10, and TNFα, following preconditioning of BM-MSCs with IL-1β. The concentration of platelet-derived growth factor BB (PDGF-BB), IL-10, and transforming growth factor-β (TGF-β) in each media was measured via immunoassay. BM-MSCs cultured in ES resulted in significant suppression of T cell proliferation (p = 0.02). Cell culture supernatant from preconditioned BM-MSCs cultured in ES had significantly higher levels of IL-6. PDGF-BB was significantly higher in APL media compared to FBS media (p = 0.016), while IL-10 was significantly higher in PPL media than ES and FBS (p = 0.04). TGF-β was highest in APL media, with a significant difference in comparison to ES media (p = 0.03). In conclusion, expansion of equine BM-MSCs in ES may enhance their immunomodulatory abilities, while PL containing media may have some inherent therapeutic potential associated with higher concentrations of growth factors. Further studies are needed to elucidate which xenogen-free supplement optimizes BM-MSC performance.
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"Comparative Analysis of the Use of Domestic Bioresorbable Collagen Membranes at the Closure of Postoperative Defects of the Oral Mucosa in an Experiment In vivo." Biointerface Research in Applied Chemistry 11, no. 2 (September 16, 2020): 9804–12. http://dx.doi.org/10.33263/briac112.98049812.
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The success of using soft tissue transplants is enough for their spreading in the clinic, but the need to cover the surface, where the transplant was taken from, can be forgotten. It can lead to long-term discomfort of the patient in real life, in some cases to complications alike bleeding; We performed the analysis of the use of different new xenogenic resorbable membranes created within our University in compare with the natural healing of oral mucosa defects in the experiment in vivo on 36 rabbits after performing of the surgical wound on the palatine side (5x5 mm). All animals were separated (divided) for 3 groups: #1 group of control and main groups #2 and #3, where we used pericardium and collagen film for covering mucous defects. We assessed the edema, hyperemia in the operation side, the pain according to animal behavior, the histological picture after animals completion of the experiment (on 3rd, 6th and 10th days); The decrease of clinical signs of inflammation in groups of collagen and pericardium films use (p<0.05) was statistically confirmed. Analysis of histologic investigation of biopsy specimens has shown the faster and massive growth of soft tissue in the donor site after application of pericardium and collagen films (p<0.05). An analysis of the experiment results allows recommending their possible use for closing the donor site after taking a free gingival graft or in the zone of postoperative wound defect in the oral mucosa in clinical oral surgery after specific clinical trials.
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Seiffert, Nicolai, Peter Tang, Eriselda Keshi, Anja Reutzel-Selke, Simon Moosburner, Hannah Everwien, Dag Wulsten, et al. "In vitro recellularization of decellularized bovine carotid arteries using human endothelial colony forming cells." Journal of Biological Engineering 15, no. 1 (April 21, 2021). http://dx.doi.org/10.1186/s13036-021-00266-5.
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Abstract Background Many patients suffering from peripheral arterial disease (PAD) are dependent on bypass surgery. However, in some patients no suitable replacements (i.e. autologous or prosthetic bypass grafts) are available. Advances have been made to develop autologous tissue engineered vascular grafts (TEVG) using endothelial colony forming cells (ECFC) obtained by peripheral blood draw in large animal trials. Clinical translation of this technique, however, still requires additional data for usability of isolated ECFC from high cardiovascular risk patients. Bovine carotid arteries (BCA) were decellularized using a combined SDS (sodium dodecyl sulfate) -free mechanical-osmotic-enzymatic-detergent approach to show the feasibility of xenogenous vessel decellularization. Decellularized BCA chips were seeded with human ECFC, isolated from a high cardiovascular risk patient group, suffering from diabetes, hypertension and/or chronic renal failure. ECFC were cultured alone or in coculture with rat or human mesenchymal stromal cells (rMSC/hMSC). Decellularized BCA chips were evaluated for biochemical, histological and mechanical properties. Successful isolation of ECFC and recellularization capabilities were analyzed by histology. Results Decellularized BCA showed retained extracellular matrix (ECM) composition and mechanical properties upon cell removal. Isolation of ECFC from the intended target group was successfully performed (80% isolation efficiency). Isolated cells showed a typical ECFC-phenotype. Upon recellularization, co-seeding of patient-isolated ECFC with rMSC/hMSC and further incubation was successful for 14 (n = 9) and 23 (n = 5) days. Reendothelialization (rMSC) and partial reendothelialization (hMSC) was achieved. Seeded cells were CD31 and vWF positive, however, human cells were detectable for up to 14 days in xenogenic cell-culture only. Seeding of ECFC without rMSC was not successful. Conclusion Using our refined decellularization process we generated easily obtainable TEVG with retained ECM- and mechanical quality, serving as a platform to develop small-diameter (< 6 mm) TEVG. ECFC isolation from the cardiovascular risk target group is possible and sufficient. Survival of diabetic ECFC appears to be highly dependent on perivascular support by rMSC/hMSC under static conditions. ECFC survival was limited to 14 days post seeding.
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Fichtel, Pascal, Malte von Bonin, Robert Kuhnert, Kristin Möbus, Martin Bornhäuser, and Manja Wobus. "Mesenchymal Stromal Cell-Derived Extracellular Vesicles Modulate Hematopoietic Stem and Progenitor Cell Viability and the Expression of Cell Cycle Regulators in an Age-dependent Manner." Frontiers in Bioengineering and Biotechnology 10 (June 1, 2022). http://dx.doi.org/10.3389/fbioe.2022.892661.
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Aging of the hematopoietic system is characterized by an expansion of hematopoietic stem and progenitor cells (HSPCs) with reduced capacity for engraftment, self-renewal, and lymphoid differentiation, resulting in myeloid-biased hematopoiesis. This process is mediated by both HSPC intrinsic and extrinsic factors, e.g., the stromal environment. A relevant cellular component of the bone marrow (BM) microenvironment are mesenchymal stromal cells (MSCs) which regulate fate and differentiation of HSPCs. The bi-directional communication with HSPCs is mediated either by direct cell-cell contacts or by extracellular vesicles (EVs) which carry bioactive substances such as small RNA, DNA, lipids and proteins. So far, the impact of MSC-derived EVs on human hematopoietic aging is poorly investigated. BM MSCs were isolated from young (n = 3, median age: 22 years) and aged (n = 3, median age: 70 years) donors and the EVs were isolated after culturing the confluent cell layer in serum-free medium for 48 h. CD34+ HSPCs were purified from peripheral blood of healthy donors (n = 3, median age: 65 years) by magnetic sorting. Nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and western blot detection of EV markers CD63, CD81 and Flotillin-1 revealed no significant differences between young and aged MSC-EVs. Interestingly, young MSCs secreted a significantly higher miRNA concentration than aged cells. However, the amount of distinct miRNAs such as miR-29a and miR-34a was significantly higher in aged MSC-EVs. HSPCs incubated with young EVs showed a significant increase in cell number and a higher viability. The expression of the tumor suppressors PTEN, a known target of mir-29a, and CDKN2A was increased in HSPCs incubated with young EVs. The clonogenic assay demonstrated a decreased colony number of CFU-GM after treatment with young EVs and an increased number of BFU-E/CFU-E after incubation with aged MSC-EVs. Xenogenic transplantation experiments showed no significant differences concerning the engraftment of lymphoid or myeloid cell compartments, but the overall human chimerism 8–16 weeks after transplantation was higher after EV treatment. In conclusion, our data suggest that HSPC characteristics such as cell cycle activity and clonogenicity can be modulated by MSC-derived EVs. Further studies have to elucidate the potential therapeutic relevance of our findings.
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Suliman, Salwa, Hassan R. W. Ali, Tommy A. Karlsen, Jerome Amiaud, Samih Mohamed-Ahmed, Pierre Layrolle, Daniela E. Costea, Jan E. Brinchmann, and Kamal Mustafa. "Impact of humanised isolation and culture conditions on stemness and osteogenic potential of bone marrow derived mesenchymal stromal cells." Scientific Reports 9, no. 1 (November 5, 2019). http://dx.doi.org/10.1038/s41598-019-52442-9.
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Abstract Therapeutic potential of human bone marrow stromal/stem cells (hBMSC) must be developed using well defined xenogenic-free conditions. hBMSC were isolated from healthy donors (n = 3) using different isolation and expansion methods. Donor I was isolated and expanded by either bone marrow directly seeded and cells expanded in 10% AB human serum (AB) +5 ng/ml fibroblast growth factor-2 (FGF2) [Direct(AB + FGFlow)] or Ammonium-Chloride-Potassium Lysing Buffer was used before the cells were expanded in 10% AB +5 ng/ml FGF-2 [ACK(AB + FGFlow)] or Lymphoprep density gradient medium was used before the cells were expanded in 10% AB +5 ng/ml FGF2 [Lympho(AB + FGFlow)] or bone marrow directly seeded and cells expanded in 10% pooled platelet lysate plasma (PL) + heparin (2 I/U/mL) [Direct(PL)]. Groups for donors II and III were: Direct(AB + FGFlow) or 10% AB +10 ng/ml FGF2 [Direct(AB + FGFhigh)] or Direct(PL). HBMSCs were assessed for viability, multi-potency, osteogenic, inflammatory response and replicative senescence in vitro after 1 and 3 weeks. Pre-selected culture conditions, Direct(AB + FGFhigh) or Direct(PL), were seeded on biphasic calcium phosphate granules and subcutaneously implanted in NOD/SCID mice. After 1 and 11 weeks, explants were analysed for inflammatory and osteogenic response at gene level and histologically. To identify implanted human cells, in situ hybridisation was performed. hBMSC from all conditions showed in vitro multi-lineage potency. hBMSCs expanded in PL expressed stemness markers in vitro at significantly higher levels. Generally, cells expanded in AB + FGF2 conditions expressed higher osteogenic markers after 1 week both in vitro and in vivo. After 11 weeks in vivo, Direct(AB + FGFhigh) formed mature ectopic bone, compared to immature mineralised tissues formed by Direct(PL) implants. Mouse responses showed a significant upregulation of IL-1α and IL-1β expression in Direct(PL). After 1 week, human cells were observed in both groups and after 11 weeks in Direct(AB + FGFhigh) only. To conclude, results showed a significant effect of the isolation methods and demonstrated a relatively consistent pattern of efficacy from all donors. A tendency of hBMSC expanded in PL to retain a more stem-like phenotype elucidates their delayed differentiation and different inflammatory expressions.