29 research outputs found
Fibrin for tissue engineering of cartilage
No full textSince the beginning of the 1990s a plethora of research approaches towards cartilage engineering for plastic and reconstructive surgery have been undertaken. However, a general standard method for generation of cartilage tissue equivalent is still lacking. The goal of this thesis is based on the project �Bavarian Research Cooperation for Tissue Engineering and Rapid Prototyping� (ForTEPro) for development of individually customized implants for facial and reconstructive surgery.
The main objective of this thesis was utilization of fibrin for tissue engineering of cartilage. Fibrin is a well-investigated medical device and has been used for over 20 years in clinical practice and surgery. Disintegration and solubility over time are taken as advantageous specific properties of fibrin glue when used as a sealant or for delivery of molecules, but for many tissue engineering based applications they represent a major problem. Therefore, specific fibrin parameters influencing gel appearance and stability were determined and a fibrin gel was developed that has a transparent appearance and is stable in size and shape under common cell culture conditions. These optimised fibrin gels showed a predominantly linear viscoelastic behaviour and withstood high mechanical loadings investigated by rheological measurements. The general suitability of the optimised fibrin gel for the use in cartilage engineering was demonstrated using primary bovine chondrocytes. Cells cultured within the gel remained round and vital, proliferated well and produced high amounts of extracellular matrix components. A minimum initial cell density was found that was required for the formation of a coherent matrix within 5 weeks. Furthermore, the optimised fibrin gels were evaluated for the suitability as surface material for culture of cells and were found to have a considerable advantageous effect on expansion of differentiated chondrocytes. The method of culturing bovine chondrocytes within the optimised fibrin gels could be successfully transferred to the use of human chondrocytes. However, generation of human cartilaginous tissue still represents a major challenge due to limited available cell number and development of smaller amounts of matrix components. Nevertheless, anabolic insulin was found to have tremendous enhancing effects on the development of extracellular matrix. In addition, combination of long-term stable fibrin gels and polymeric scaffolds was also investigated. Injection of a cell-fibrin suspension into newly developed polycaprolactone-based polyurethane scaffolds resulted in formation of adequate cartilaginous tissue, at the same time providing an added stability for the delicate process of implantation and for mechanical loading in vivo. Finally, a polycaprolactone-based scaffold in the shape of the cartilage part of an adult human ear was successfully seeded with chondrocytes suspended in fibrin gel. Though previous studies reported problems with construct shrinkage and deformation, in this experiment, construct size and shape was highly preserved within the culture period. In a further study, the potential of the composite constructs was investigated in detailed in vivo experiments. Small constructs implanted into the back of nude mice for up to 6 months showed formation of adequate new cartilaginous tissue. Remarkably, in vitro pre-culture of these constructs was demonstrated to have strong enhancing effects on further in vivo tissue development. Interestingly, a short in vitro pre-culture period of 1 week elicited the same results as a longer period of four weeks. This observation may have advantageous implications for future applications in clinical practise.
In conclusion, within this thesis, a long-term stable fibrin gel was developed. The potential of this fibrin gel for application in cartilage engineering was demonstrated, using either bovine or delicate human chondrocytes. In combination with a solid polycaprolactone-based scaffold, a prototype of an individually customized cartilaginous construct in the shape of the external part of a human ear was generated in vitro. Disc-shaped composite constructs showed satisfying long-term development and maintenance of engineered cartilage after subcutaneous implantation in nude mice. In future, this system may result in fully functional individually shaped implants for cartilage regeneration
Immunoinformatics of Placental Malaria Vaccine Development
No full textMalaria er en infektionssygdom, forårsaget af en protozoisk parasit af slægten Plasmodium. Parasitten overføres af hunmyg af arten Anopheles. WHO estimerer at der var 207 millioner tilfælde af malaria i 2012, hvoraf 627.000 var fatale.Mennesker, som bor i malariaendemiske områder, erhverver gradvist immunitet, med stigende antal infektioner. Placental malaria (PM) forårsages af P. falciparum, som afsondres i moderkagen hos gravide kvinder. Dette skyldes tilstedeværelsen af nye receptorer i moderkagen. Det estimeres at der hvert år dør 200.000 spædbørn af PM. I 2004 blev det specifikke protein, som er ansvarlig for patogenesen af PM, identificeret som P. falciparum Erythrocyt Membran Protein 1 (Pf EMP1) varianten VAR2CSA. VAR2CSA er den førende kandidat til en vaccine mod PM. Denne ph.d. afhandling er inddelt i 4 hoveddele, hvor del I forsyner læseren med en introduktion til og baggrund for emnerne, indeholdt i afhandlingen. Del II præsenterer den første artikel: ”SigniSite: Identifikation af aminosyrerest-niveau genotype-fænotype korrelationer i multiple sekvens alignment af proteiner”. SigniSite er baseret på en ikke-parametrisk statistisk evaluering af den positionelle aminosyrerestfordeling i et multipelt sekvensalignment (MSA), hvorved aminosyrens associering med MSA fænotypen kvantificeres. Vi fandt at SigniSite var i stand til at performe bedre end den nyeste sammenlignelige metode. Ydermere adresserer del II udfordringen med at kontrollere Dissertation advisor: Prof. Ole Lund Leon Eyrich Jessen sandsynligheden for fejl af type I og type II i multiple testscenarier. Sluttelig anvendes SigniSite til at analysere MHCI:peptide bindingsinteraktionerne. Del III præsenterer den anden artikel: ”Indsigt i den antigeniske diversitet af VAR2CSA-DBLε domænet fra multiple Plasmodium falciparum placentale isolater”.Dataene bestod af 70 VAR2CSA-DBL5ε sekvenser, hver især associeret med et sæt af fænotyper. Immunitet mod PM erhverves gradvist, hvorfor såfremt et givet sekvensmotiv kan fænotype korreleres, så er dette motiv muligvis involveret i VAR2CSA immunogenicitet. Motiver, som definerer VAR2CSA immunogenecitet er naturligvis interessant i kontekst med vaccineudvikling.Motivet ’TFKNI’ korrelerede med barnets fødselsvægt. Del IV præsenterer udviklingen og anvendelsen af to metoder til analyse af høj-produktionsdata fra en ny Høj Densitets Peptid Mikroarray (HDPMa) chip teknologi. HDPMa chippen blev efterfølgende anvendt til opsporing af lineære b-celle epitoper.Peptiderne ’GMDEFKNTFKNIKE’ og ’SCGSARTMKRGYKNDNYELCKYC’ blev identificeret som lineære b-celle epitoper. Den sidste af de to, blev efterfølgende eksperimentelt fundet til at være høj-immunogent, men ikke i stand til at blokere VAR2CSA:receptor interaktionen. I sammendrag, centrerer arbejdet, som beskrives i denne ph.d. afhandling sig om udviklingen og anvendelsen af bioinformatiske værktøjer til in silico analyse af VAR2CSA, med særlig vægt på statistisk metodologi. Det er forfatterens håb at værktøjerne udviklet og anvendt i denne ph.d. afhandling, kan tjene som udgangspunkt for, eller blot bidrage til, videre forskning og udvikling i feltet for placental malaria vaccineudvikling.Malaria is an infectious disease caused by a protozoan parasite of the genus Plasmodium, which is transferred by female Anopheles mosquitos. WHO estimates that in 2012 there were 207 million cases of malaria, of which 627,000 were fatal. People living in malaria-endemic areas, gradually acquire immunity with multiple infections. Placental malaria (PM) is caused by P. falciparum sequestering in the placenta of pregnant women due to the presence of novel receptors in the placenta. An estimated 200,000 infants die a year as a result of PM. In 2004 the specific protein responsible for the pathogenesis of PM was identified as the P. falciparum Erythrocyte Membrane Protein 1 (Pf EMP1) variant VAR2CSA. VAR2CSA is the leading candidate for a vaccine against PM.The thesis is divided into 4 parts, where part I provide the reader with an introduction and background for the subjects covered in the thesis. Part II presents the first paper: ”SigniSite: Identification of residue-level genotype-phenotype correlations in protein multiple sequence alignments”. SigniSite is based on a non-parametric statistical evaluation of the positional distribution of amino acid residues in a multiple sequence alignment (MSA), thereby quantifying residue association to MSA phenotype. SigniSite was found to outperform comparable state-of-the-art methods. Furthermore part II addresses the issue of controlling type I and type II error probabilities in multiple testing scenarios and lastly the Dissertation advisor: Prof. Ole Lund Leon Eyrich Jessen analysis of the MHCI:peptide binding interaction by application of the SigniSite method. Part III presents the second paper: ”Insight into Antigenic Diversity of VAR2CSA-DBL5ε Domain from Multiple Plasmodium falciparum Placental Isolates”. The data consisted of 70 VAR2CSA-DBL5ε sequences each with associated phenotypes. Immunity towards PM is gradually acquired, therefore if a given sequence motif can be phenotype-correlated then the motif may be involved in VAR2CSA immunogenecity. Motifs defining VAR2CSA immunogenecity are naturally interesting in vaccine development context. The motif ’TFKNI’ was found to be correlated with the birth weight of the child. Part IV presents the development of two methods for analysis of high-throughput data from a novel High Density Peptide Microarray (HDPMa) chip technology.Subsequently the HDPMa chip is applied for the discovery of linear B-cell VAR2CSA epitopes. Peptides ’GMDEFKNTFKNIKE’ and ’SCGSARTMKRGYKNDNYELCKYC’ were identified as linear B-cell epitopes. The latter subsequently experimentally found to be highly immunogenic, but not capable of blocking VAR2CSA:receptor interaction. In summary, the work described in this thesis centres around the development and application of bioinformatics tools for in silico analysis of VAR2CSA, with an emphasis on statistical methodology. It is the hope of the author that the tools, developed, presented and applied in this thesis, may serve as an offset for further research and development in the field of placental malaria vaccine development
Effects of interleukin-4 on glycosaminoglycan content and matrix metalloproteinase expression in engineered cartilage
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Sustained Peptide and Protein Release from Lipid Microparticles Prepared by Spray Congealing
No full textCombination of long-term stable fibrin gels and polycaprolactone-based scaffolds for cartilage engineering
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