Articular cartilage defects are still one of the major challenges in orthopedic and trauma surgery. Today, autologous chondrocyte transplantation (ACT), as a cell-based therapy, is an established procedure. However, one major limitation of this technique is the loss of the chondrogenic phenotype during expansion. Human mesenchymal stem cells (hMSCs) have an extensive proliferation potential and the capacity to differentiate into chondrocytes when maintained under specific conditions. They are therefore considered as candidate cells for tissue engineering approaches of functional cartilage tissue substitutes. First in this study, hMSCs were embedded in a collagen type I hydrogel to evaluate the cartilaginous construct in vitro. HMSC collagen hydrogels cultivated in different culture media showed always a marked contraction, most pronounced in chondrogenic differentiation medium supplemented with TGF-ß1. After stimulation with chondrogenic factors (dexamethasone and TGF-ß1) hMSCs were able to undergo chondrogenesis when embedded in the collagen type I hydrogel, as evaluated by the temporal induction of cartilage-specific gene expression. Furthermore, the cells showed a chondrocyte-like appearance and were homogeneously distributed within a proteoglycan- and collagen type II-rich extracellular matrix, except a small area in the center of the constructs. In this study, chondrogenic differentiation could not be realized with every hMSC preparation. With the improvement of the culture conditions, e.g. the use of a different FBS lot in the gel fabrication process, a higher amount of cartilage-specific matrix deposition could be achieved. Nevertheless, the large variations in the differentiation capacity display the high donor-to-donor variability influencing the development of a cartilaginous construct. Taken together, the results demonstrate that the collagen type I hydrogel is a suitable carrier matrix for hMSC-based cartilage regeneration therapies which present a promising future alternative to ACT. Second, to further improve the quality of tissue-engineered cartilaginous constructs, mechanical stimulation in specific bioreactor systems are often employed. In this study, the effects of mechanical loading on hMSC differentiation have been examined. HMSC collagen hydrogels were cultured in a defined chondrogenic differentiation medium without TGF-ß1 and subjected to a combined mechanical stimulation protocol, consisting of perfusion and cyclic uniaxial compression. Bioreactor cultivation neither affected overall cell viability nor the cell number in collagen hydrogels. Compared with non-loaded controls, mechanical loading promoted the gene expression of COMP and biglycan and induced an up-regulation of matrix metalloproteinase 3. These results circumstantiate that hMSCs are sensitive to mechanical forces, but their differentiation to chondrocytes could not be induced. Further studies are needed to identify the specific metabolic pathways which are altered by mechanical stimulation. Third, for the development of new cell-based therapies for articular cartilage repair, a reliable cell monitoring technique is required to track the cells in vivo non-invasively and repeatedly. This study aimed at analyzing systematically the performance and biological impact of a simple and efficient labeling protocol for hMSCs. Very small superparamagnetic iron oxide particles (VSOPs) were used as magnetic resonance (MR) contrast agent. Iron uptake was confirmed histologically with prussian blue staining and quantified by mass spectrometry. Compared with unlabeled cells, VSOP-labeling did neither influence significantly the viability nor the proliferation potential of hMSCs. Furthermore, iron incorporation did not affect the differentiation capacity of hMSCs. The efficiency of the labeling protocol was assessed with high resolution MR imaging at 11.7 Tesla. VSOP-labeled hMSCs were visualized in a collagen type I hydrogel indicated by distinct hypointense spots in the MR images, resulting from an iron specific loss of signal intensity. This was confirmed by prussian blue staining. In summary, this labeling technique has great potential to visualize hMSCs and track their migration after transplantation for articular cartilage repair with MR imaging. / Gelenkknorpeldefekte stellen immer noch eine der großen Herausforderungen in der Orthopädie und Unfallchirurgie dar. Als zellbasiertes Verfahren ist die Autologe Chondrozytentransplantation (ACT) heute in der klinischen Routine etabliert. Ein großer Nachteil dieser Methode ist jedoch der Verlust des chondrozytären Phänotyps während der Expansion der Zellen. Humane mesenchymale Stammzellen (hMSZ) verfügen über ein ausgeprägtes Proliferationspotential und besitzen die Fähigkeit, unter spezifischen Bedingungen zu Knorpelzellen zu differenzieren. Sie werden daher als alternative Zellen für das Tissue Engineering von funktionellem Knorpelersatzgewebe in Betracht gezogen. In der vorliegenden Arbeit wurden erstens hMSZ in ein Kollagen Typ I Hydrogel eingebracht und zunächst der Grad der chondrogenen Zelldifferenzierung im Konstrukt evaluiert. HMSZ-Kollagenhydrogele zeigten in allen Kultivierungsmedien eine deutliche Kontraktion, welche am stärksten im chondrogenen Differenzierungsmedium unter Zugabe von TGF-ß1 ausgeprägt war. Nach Stimulation mit chondrogenen Faktoren (Dexamethason und TGF-ß1) differenzierten hMSZ zu Knorpelzellen, nachgewiesen durch die Induktion von knorpelspezifischer Genexpression. Die Zellen wiesen eine Chondrozyten-ähnliche Morphologie auf und waren bis auf einen kleinen Bereich in der Mitte des Konstrukts homogen in einer Proteoglykan- und Kollagen Typ II-haltigen extrazellulären Matrix verteilt. Eine chondrogene Differenzierung konnte in der vorliegenden Arbeit jedoch nicht mit jeder hMSZ-Präparation realisiert werden. Durch die Verbesserung der Kulturbedingungen, z.B. durch die Verwendung einer anderen Serumcharge im Gelherstellungsprozess, konnte eine Steigerung der knorpelspezifischen Matrixsynthese erzielt werden. Nichtsdestotrotz spiegeln die großen Schwankungen in der Differenzierungskapazität die hohe Variabilität zwischen verschiedenen Spendern wider, welche die Entwicklung eines knorpelartigen Gewebes beeinflussen. Zusammengefasst zeigen die Ergebnisse, dass das Kollagen Typ I Hydrogel eine geeignete Trägermatrix für hMSZ darstellt, um in Stammzell-basierten Knorpelregenerationstherapien zukünftig als vielversprechende Alternative zur ACT eingesetzt zu werden. Um die Qualität eines in vitro generierten knorpelartigen Gewebes weiter zu verbessern, wird häufig eine mechanische Stimulation in spezifischen Bioreaktorsystemen durchgeführt. In der vorliegenden Arbeit wurden daher zweitens die Effekte von mechanischer Belastung auf die Differenzierung von hMSZ untersucht. HMSZ-Kollagenhydrogele wurden im chondrogenen Differenzierungsmedium ohne TGF-ß1 kultiviert und einem kombinierten mechanischen Stimulationsprotokoll, bestehend aus Perfusion und zyklischer uniaxialer Kompression, ausgesetzt. Die Kultivierung im Bioreaktor hatte weder einen Einfluss auf die Zellvitalität noch auf die Anzahl der Zellen im Kollagen Typ I Hydrogel. Die mechanische Beeinflussung steigerte im Vergleich mit den unbelasteten Kontrollgelen die Genexpression von COMP und Biglykan und führte zu einer Hochregulation von Matrix Metalloproteinase 3. Diese Ergebnisse belegen, dass hMSZ mechanosensitiv sind, jedoch konnte keine Differenzierung zu Knorpelzellen induziert werden. Hierfür sind weitere Studien notwendig, um spezifische Stoffwechselwege zu identifizieren, welche durch die mechanische Stimulation beeinflusst werden. Drittens, für die Entwicklung von neuen zellbasierten Therapien für die Gelenkknorpelrekonstruktion ist eine zuverlässige Bildgebung auf zellulärer Ebene erforderlich, um die Zellen in vivo wiederholt nicht invasiv zu detektieren. Die vorliegende Arbeit hatte zum Ziel, systematisch die Effizienz und die biologischen Auswirkungen einer einfachen und dauerhaften Markierung für hMSZ zu untersuchen. Superparamagnetische Eisenoxidnanopartikel (VSOPs), ein Magnetresonanz (MR)-Kontrastmittel, wurden für die Markierung eingesetzt. Die Aufnahme der Eisenoxidnanopartikel wurde histologisch mittels eisenspezifischer Berliner-Blau-Färbung nachgewiesen und durch Massenspektroskopie quantifiziert. Im Vergleich zu unmarkierten Zellen beeinträchtigte die VSOP-Markierung weder die Vitalität noch das Proliferationspotential der hMSZ. Weiterhin war durch die Aufnahme der Eisenoxidnanopartikel keine Beeinflussung der Differenzierungskapazität der hMSZ zu verzeichnen. Die Effizienz der Zellmarkierung wurde mittels hochauflösender MR-Bildgebung bei 11,7 Tesla beurteilt. VSOP-markierte hMSZ im Kollagen Typ I Hydrogel erschienen als hypointense Punkte in den MR-Bildern, hervorgerufen durch die typische, VSOP-bedingte Signalauslöschung. Histologische Untersuchungen dieser Konstrukte bestätigten die MR-Ergebnisse. Zusammenfassend lässt sich festhalten, dass diese Zellmarkierungsmethode in Verbindung mit der MR-Bildgebung über das Potential verfügt, nach einer Gelenkknorpelrekonstruktion Aufschluss über die Lokalisation und Migration der transplantierten hMSZ zu liefern.

Für die Rekonstruktion von Gelenkknorpeldefekten des Kniegelenkes in Folge eines Traumas oder einer Osteochondrosis dissecans (OD) stehen verschiedene operative Verfahren zur Verfügung. Die Autologe Chondrozytentransplantation (ACT) hat sich als zuverlässiges Rekonstruktionsverfahren erwiesen. In der vorliegenden Arbeit wurde eine prospektive Fallseriestudie für eine neue Form der ACT mit einem Kollagen I Hydrogel (CaReS-Technologie) durchgeführt. Die Vorteile der Technologie liegen zum Einen darin, dass sich die Zellen homogen im Gel verteilen und zum Anderen, dass die Zellen unmittelbar nach dem Herauslösen aus dem Gelenkknorpel in das Gel eingebracht werden und dadurch eine geringere Dedifferenzierung der Chondrozyten stattfindet. Von März 2003 bis Ende 2006 wurden 29 Patienten in die Studie eingeschlossen. Die Ein- und Ausschlusskriterien erfüllten die Kriterien der Arbeitsgruppe ACT und Tissue Engineering der Deutschen Gesellschaft für Orthopädie und Unfallchirurgie. Die Eingangs- und Nachuntersuchungsbögen wurden an die IKDC Form 2000 angelehnt. Insgesamt zeigte sich ein signifikanter Anstieg des IKDC Scores im mittleren follow-up von 30,7 Monaten von 47,3 auf 74,9 bei den 29 Patienten. Bei Aufschlüsselung der Patienten bzgl. Diagnose, Defektgröße, Lokalisation und Defektanzahl zeigte sich bei den Behandlungsgruppen OD, Trauma/degenerativ, > 4 cm2, mediale Femurkondyle und Einzeldefekte eine signifikante Zunahme des IKDC Scores im zeitlichen Verlauf. Der postoperative Schmerz zeigte einhergehend mit dem Anstieg des IKDC Scores eine signifikante Abnahme der Schmerzintensität in den Behandlungsgruppen OD, Trauma/degenerativ, > 4 cm2, mediale Femurkondyle und Einzeldefekte. Nachgewiesen wurde ebenfalls ein Anstieg des SF36 Scores, der den gegenwärtigen Gesundheitszustand sowohl körperlich als auch psychisch beurteilt. Zusammen mit einer globalen Patientenzufriedenheit von 80% und einem IKDC Funktionsstatus von I und II bei 77% der Patienten spiegeln die gewonnenen Daten die Ergebnisse der klassischen ACT bzw. anderer matrixgekoppelten Verfahren wieder. Die CaReS-Technologie stellt somit ein gleichwertiges Verfahren zu den bisher auf dem Markt befindlichen Techniken der ACT dar. / For the reconstruction of articular cartilage defects of the knee resulting from osteochondritis dissecans (OD) or trauma different surgical techgniques are available. Autologous chondrocyte implantation (ACI) has proven to be a reliable reconstructive technique. In this work, we have performed a prospective case control study using a new form of ACI with a collagen type I hydrogel (CaReS-technology). The advantages of the technology are that the cells can be distributed homogeneously within the hydrogel and immidiatly after their release from the cartilage they are brought into the gel, resulting in less dedifferentiation of the chondrocytes. From March 2003 to the end of 2006 we enrolled 29 patients in this study. The inclusion and exclusion criteria were adopted from the criteria of the working group for ACT and Tissue Engineereing of the German Society for Orthopaedic and Trauma Surgery. The inital clinical and follow-up examinations were performed according to the IKDC form 2000. In general, we found a significant increase of the IKDC score at the follow-up of 30.7 months from 47.3 to 74.9 of the 29 patients. When seperating the patients by diagnosis, defect size, defect location and defect number we found in the groups with OD, trauma/degenerativ, > 4 cm2, medial condyle and single defects a significant increase of the IKDC score over the follow-up period. The postoperative pain showed accoring to the increased IKDC score a significant decrease of the pain intensity in the groups with OD, trauma/degenerativ, > 4 cm2, medial condyle and single defects. We also found an increase of the SF36 score, which describes the current physical and mental health status. Together with a global patient satisfaction of 80% and an IKDC functional status of I and II in 77% of the patients our data reflect the results of the classical ACI, as well as other matrix-based technologies. Therefore, the CaReS-technology is comparable to other technologies which are currently on the market.

Student Number : 9501020M - M Sc dissertation - School of Clinical Medicine - Faculty of Health Sciences / An exciting and novel concept of tissue engineering and morphogenesis is the generation of bone by the implantation of smart biomaterials that in their own right can induce a desired and specific morphogenetic response from the host tissues without the addition of exogenously applied bone morphogenetic and osteogenic proteins.

Estima-se, que, no Brasil, ocorram cerca de um milhão de vítimas de queimaduras por ano, e mesmo com a dinâmica de inovações na área da saúde, a reparação deste tipo de lesão tecidual, permanece um grande desafio. Os queimados tendem a contrair infecções sistêmicas, as quais poderão levar a óbito, se não houver o tratamento adequado ao paciente. Desta forma, são necessários cuidados extremos nas etapas que envolvem este complexo reparo tissular. Diante das dificuldades na substituição ou regeneração de órgãos ou tecidos lesionados, surgiu um campo interdisciplinar chamado de engenharia de tecidos, com foco no estudo para o desenvolvimento de suportes tridimensionais, constituídos de materiais sintéticos ou naturais, onde são cultivadas células do próprio paciente, para posteriormente serem reinseridas reparando tecidos ou substituindo órgãos por inteiro. A quitosana é um dos biopolímeros mais utilizados hoje na área de engenharia de tecidos, devido a sua capacidade de agir de forma significativa nas três fases que envolvem a cicatrização de queimaduras, sendo elas: a fase inflamatória, a fase proliferativa e a fase reparadora, e por sua alta ação bacteriostática e fungistática. Diante das propriedades já existentes da quitosana, o objetivo desta pesquisa foi o estudo para intensificá-las, através do aumento do seu grau de desacetilação e modificando-a para uma escala nanométrica aumentando assim sua área superficial. Para isso, a quitosana foi submetida a meio altamente alcalino com variação de temperatura e variação do tempo de reação, utilizando a ferramenta estatística fatorial completo 23. Após a obtenção das amostras desacetiladas, foi verificado, através dos espectros obtidos por espectroscopia na região do infravermelho, que os maiores valores de grau de desacetilação ocorreram utilizando os níveis máximos em todos os fatores envolvidos na reação. Para analisar a cinética da reação e confirmar as informações obtidas do fatorial 23, foi feito um novo planejamento fatorial 22, fixando o tempo de seis horas de reação, e no decorrer deste tempo foram retiradas onze alíquotas, para análise de seus graus de desacetilação (GD). O padrão de resultados dos experimentos permitiu a aplicação de um modelo matemático que representou a realidade do que ocorreu durante a reação, sendo este o modelo do núcleo não reagido. Posteriormente, a quitosana com alto grau de desacetilação foi submetida ao método de ultrassom e pelas análises do diâmetro das partículas, potencial zeta e índice de polidispersão, foi possível verificar que a quitosana após ser submetida ao ultrassom e no pH adequado, foi possível atingir partículas em escala nanométrica. / It is estimated that in Brazil about one million burn victims occur per year, and even with the dynamics of innovations in the health area, the repair of this type of tissue injury, remains a great challenge. Burns tend to contract systemic infections, which can lead to death if the patient is not adequately treated. In this way, extreme care is required in the steps involved in this complex tissue repair. Faced with difficulties in the replacement or regeneration of injured organs or tissues, an interdisciplinary field called tissue engineering has emerged, focusing on the study for the development of three-dimensional supports, consisting of synthetic or natural materials, where the patient\'s own cells are cultured, subsequently reinserted by repairing tissues or replacing whole organs. Chitosan is one of the most widely used biopolymers nowadays in the field of tissue engineering, due to its capacity to act in a significant way in the three phases that involve the healing of burns, namely: inflammatory phase, proliferative phase and repair phase, and for its high bacteriostatic and fungiostatic action. In view of the existing properties of chitosan, the objective of this research was to intensify them by increasing its degree of deacetylation and modifying it to a gauge scale, thus increasing its surface area. For this, chitosan was submitted to a highly alkaline medium with temperature variation and reaction time variation, using the complete factorial statistical tool 23. After obtaining the deacetylated samples, it was verified by spectroscopy in the infrared region, that the highest values of deacetylation degree occurred after using the maximum levels in all factors involved in the reaction. In order to analyze the kinetics of the reaction and to confirm the information obtained from factorial 23, a new 22 factorial design was made, fixing the time of six hours of reaction, during which eleven aliquots were taken for analysis of their degree of desacetylation (GD). The pattern of results of the experiments allowed the application of a mathematical model that represented the reality of what occurred during the reaction, being this the model of the shrinking core model. Subsequently, the chitosan with a high degree of deacetylation was subjected to the ultrasound method and the analysis of particle diameter, zeta potential and polydispersion index allowed to verify that chitosan after being submitted to ultrasound at the appropriate pH achieved particles in nanometer scale.

A utilização e o desenvolvimento de biomateriais para a regeneração tecidual são de grande importância, principalmente para a área médica e odontológica. Matrizes de colágeno derivadas de tecidos de origem animal são utilizadas devido o colágeno apresentar características como biodegradabilidade e biocompatibilidade. Essas matrizes podem ser obtidas a partir de várias fontes, sendo uma delas o pericárdio porcino, que apresenta vantagens como grande disponibilidade, baixo custo, fácil obtenção e possibilidade de sofrer modificações químicas. Além disso, tecidos de origem suína são muito similares aos tecidos humanos, podendo ser utilizados para a produção de biomateriais para a regeneração de tecido mole. Este trabalho teve como objetivo a preparação de membranas acelulares derivadas de pericárdio porcino por hidrólise alcalina em diferentes tempos, para posterior utilização em engenharia de tecido. As membranas de colágeno foram obtidas por hidrólise alcalina de pericárdio porcino durante 4, 8, 12 e 24 h e caracterizadas por análise histológica, microscopia eletrônica de varredura (MEV), avaliação da citoxicidade in vitro, estabilidade biológica in vitro (colagenase), titulação potenciométrica, porcentagem de absorção de água, calorimetria exploratória diferencial (DSC), termogravimetria (TG), análise térmica dinâmico-mecânica (DMTA) e ensaios de tração. A análise histológica mostrou que após 4h de hidrólise as células foram removidas das membranas. A avaliação da citotoxicidade in vitro mostrou que as membranas preparadas neste trabalho não são citotóxicas. Os ensaios de estabilidade biológica in vitro por colagenase mostraram que as membranas hidrolisadas degradaram mais rapidamente que a não hidrolisada e, quando comparadas com matrizes derivadas de pericárdio bovino, as derivadas de pericárdio porcino foram mais resistentes à degradação por colagenase. A titulação potenciométrica possibilitou determinar o número de grupos carboxílicos das membranas e o incremento desses grupos por molécula de colágeno. Os resultados mostraram que houve um aumento no número de grupos carboxílicos tituláveis nas membranas hidrolisadas e, consequentemente, houve um aumento do número de cargas negativas incorporadas na molécula de colágeno. As membranas hidrolisadas apresentaram uma maior absorção de água, uma diminuição das temperaturas de desnaturaçãoe e menor estabilidade térmica em função do aumento do tempo de hidrólise. Os ensaios de tração mostraram que após a hidrólise alcalina as membranas apresentaram maiores valores de resistência à tração e que a deformação é dependente do tempo de hidrólise alcalina. Esses resultados mostraram que a preparação de membranas de colágeno derivadas de pericárdio porcino com diferentes tempos de hidrólise alcalina é um procedimento viável para ser utilizado na produção de biomateriais para engenharia de tecido. / The use and development of biomaterials for tissue regeneration are of great importance, especially for medical and dental care. Collagen matrices derived from animal tissues are widely used because collagen has characteristics such as biodegradability and biocompatibility. These matrices can be obtained from various sources, such as porcine pericardium, which is a tissue that can be used due its low cost, wide availability and because it can be chemically modified. Besides, porcine tissues are very similar to human tissue and can be used to produce biomaterials for soft tissue regeneration. The aim of this study was the preparation and characterization acellular membranes by alkaline hydrolysis of porcine pericardium. Membranes were characterized by histological analysis, scanning electron microscopy (SEM), in vitro cytotoxicity evaluation, in vitro biological stability (collagenase), potentiometric titration, water absorption percentage, differential scanning calorimetry (DSC), thermogravimetry (TG), dynamic mechanical thermal analysis (DMTA) and tensile tests. Histological analysis showed that after 4h of hydrolysis, cells were totally removed from matrices. In vitro cytotoxicity showed that all matrices prepared in this work are not cytotoxic. In vitro biological stability tests (collagenase) showed that the hydrolyzed membranes degraded more quickly than the non hydrolized matrix and more resistant to collagenase degradation when compared to matrices derived from bovine pericardium. The potentiometric titration allowed the determination carboxylic groups and the increase of these groups per collagen molecule. Hydrolyzed matrices had an increase in water absorption, a decrease in denaturation temperature and a small decrease in thermal stability with the increase of hydrolysis time. Tensile tests showed that after alkaline hydrolysis matrices showed higher tensile strength and the deformation was independent of the time of alkaline hydrolysis. These results showed that the preparation of collagen biological matrices derived from porcine pericardium with different times of alkaline hydrolysis is a viable procedure to be subsequently used in the production of biomaterials for tissue engineering.

Cellular activities are composite responses to stimuli from the surroundings. Materials for biological applications, therefore, must be designed with care such that undesired interactions between cells and the materials will not be elicited while cellular responses that are beneficial to the dedicated applications are promoted. Efforts have been made to construct such materials based on both synthetic polymers and natural polymers including poly(ethylene glycol) (PEG) and proteins. In particular, recombinant proteins have drawn great interest for their similar biocompatibility to natural proteins and the uniformity of material properties that is found in manufacturing of synthetic polymers. Recombinant proteins are designed at the DNA level, which allows precise control over the translated protein sequence. By assembling encoded DNA sequences of amino acids with desired functional groups or protein domains conferring desired functionalities, a recombinant protein-based material can be tailored. In this dissertation, works toward developing functional biomaterials based on both synthetic polymers and recombinant proteins are presented.<br>The first part of this thesis encompasses the development of a new thiol-based crosslinking approach to achieve independent control over degradability and mechanical properties of a hydrogel system. Thiol chemistry was chosen as the focus here because it can easily be incorporated into recombinant protein designs by inserting cysteine residues. In addition, the low frequency of cysteine residues in natural proteins can reduce unwanted reactions between the hydrogel material and encapsulated biomolecules or cells. We utilized divinyl sulfone (DVS) to form thioether crosslinking through thiol-ene addition and ferric ethylenediaminetetraacetic acid (ferric EDTA) to make disulfide crosslinking via thiol oxidation. By controlling the ratio between the non-reducible thioether bonds to reducible disulfide bonds, hydrogels with similar mechanical properties can be made with different degradability in reducing conditions. Accelerated degradation and increased release of encapsulated dextran was observed in response to an extracellular reducing condition. Good viability of encapsulated fibroblasts also suggested high cytocompatibility of the crosslinking approach. This work demonstrated the potential of thiol crosslinking by DVS and ferric EDTA for making redox-responsive drug delivery vehicles and tissue engineering scaffolds.<br>In the second part, we developed protein adhesives using thiol- or catechol-based adhesion. Every year more than 310 million surgeries are performed around the world, and more than 50% of these surgeries used sutures or staples for wound closure. Surgical sealants or adhesive can be applied together with sutures and staples to mitigate the risk of infection. Protein-based adhesives could have better biocompatibility than synthetic polymer-based adhesives and have the potential of providing biochemical cues for cellular responses. Many adhesive proteins have been found in nature. Among them, mussel adhesive proteins have been actively studied for their outstanding underwater adhesion. The capability of being able to cure in a wet environment is critical for an ideal surgical sealant and adhesive. Mussels uses both thiols and a catechol, 3, 4-dihydroxyphenylalanine (DOPA), to achieve underwater adhesion. Inspired by mussel adhesive proteins and modular recombinant design, we developed two proteins harboring thiol or DOPA groups with highly similar amino acid sequences. The adhesion performance, including curing kinetics, adhesion strength, and cytocompatibility, were compared between the two proteins. The similarity in the protein sequences allows us to focus on the performance difference between thiol- and DOPA-based adhesion. We also showed that a synergistic increase in the adhesion strength can be achieved when the two proteins are combined. This increase indicates a cross-reaction between thiol and DOPA groups. Our results provide insights into selecting the chemistry for designing adhesives based on the needs of the applications.<br>In the last part, we studied the lower critical solution temperature (LCST) behavior of elastin-like polypeptides (ELPs) with a series of ELPs with rationally designed charge distributions and chain lengths. The LCST behavior of ELPs are controlled by multiple factors including the amino acid composition, ELP chain length, protein concentration, salt identity, salt concentration, and pH of the solution. Fusion of other non-ELP recombinant protein domains to ELPs have also been shown to influence the LCST behavior of the fusion ELP protein. Inspired by this effect, we explored the use of short non-ELP sequences as a new way to tailor the LCST behavior of ELP-based proteins. We designed the non-ELP and the ELP sequences with different pH-dependent charge states and showed that pH sensitivity was introduced to the LCST behavior by electrostatic and hydrophobic interactions between the non-ELP and ELP sequences. The electrostatic interactions can be shielded by the ionic strength in the protein solution. The pH sensitivity was introduced by the non-ELP sequences, and this sensitivity decreased when the relative length of the ELP domain increased. We also found that the hydrophobicity of the non-ELP sequences changes the interactions between the proteins and Hofmeister ions in solution. Our results demonstrated the potential of using non-ELP sequences as a new factor in controlling the LCST behavior of ELP proteins.<br><br>

A bioengenharia de tecidos baseia-se no uso de moléculas bioativas, células-tronco e biomateriais para reparação de tecidos e/ou órgãos. Biomateriais podem ser classificados de acordo com sua origem em sintéticos ou biológicos. Biomateriais biológicos podem ser produzidos por decelularização, que visa a remoção de células da matriz extracelular (MEC), a qual deve manter sua integridade química e física. Placentas são órgãos de grande interesse na bioengenharia de tecidos visto que são descartadas após o parto e possuem grande volume de matriz extracelular. Métodos de decelularização podem ser classificados em químicos, físicos e enzimáticos. Todos conhecidamente causam alterações na MEC, sendo que a associação deles é comumente utilizada. Este trabalho comparou diferentes protocolos e estabeleceu um método mais favorável para a decelularização de placentas caninas, visando a produção de um biomaterial para futuras aplicações clínicas. Inicialmente ambas as porções - materna e fetal - das placentas foram submetidas à 10 protocolos, que avaliaram variáveis como concentração e tempo de incubação em detergentes, diferentes gradientes de temperatura e a influência da perfusão versus imersão das soluções, na MEC remanescente. Com base na transparência do tecido e na ausência de núcleo celular em cortes histológicos, dois protocolos foram selecionados (I e II). Além dos critérios já mencionados, ambos os protocolos foram comparados quanto à quantidade de DNA remanescente na MEC decelularizada e à permanência e distribuição de algumas das proteínas da matriz. O detergente SDS foi o mais eficaz na remoção de células, embora não tenha sido suficiente para promover uma decelularização tecidual completa. O congelamento prévio das placentas requereu um maior tempo de incubação posterior das amostras nos distintos detergentes. Ambos métodos de perfusão e imersão foram eficazes na remoção das células, embora grande concentração de proteínas do citoesqueleto tenham permanecido retidas na matriz. As amostras processadas pelo protocolo I (SDS 1%, 5mM EDTA &#43; 50mM TRIS &#43; 0,5% antibiótico, e Triton X-100 1%) apresentaram maior preservação da organização estrutural da MEC quando comparadas àquelas processadas de acordo com o protocolo II (que diferiu do anterior pela utilização de solução contendo 0,05% tripsina ao invés de 50mM TRIS), esse último método entretanto foi o que melhor removeu as células das placentas, conforme observado em lâminas histológicas e demonstrado pela menor concentração de DNA. Tanto as porções materna quanto fetal submetidas à ambos protocolos, mantiveram as proteínas laminina, fibronectina e colágeno tipo I. O colágeno tipo III foi observado somente na porção fetal. Conclui-se que o protocolo II foi o mais eficaz no processo de decelularização de placentas caninas tendo promovido a remoção do conteúdo celular e diminuição da concentração de DNA na MEC remanescente. No entanto é necessário otimizar o tempo de incubação das placentas em soluções enzimáticas visando maior conservação do arranjo da matriz decelularizada. A análise da capacidade da MEC decelularizada por tal método para ser utilizada em bioengenharia de tecidos ainda deve ser avaliada in vitro e in vivo / Tissue engineering is based on the use of bioactive molecules, stem cells and biomaterials to repair tissues and/organs. Biomaterials can be classified according to their origin in synthetic or biological. Biological biomaterials can be produced by decellularization wich aims at removing cells from the extracellular matrix (ECM), while maintain its chemical and physical integrity. Placentas are organs of great interest in tissue engineering due to the fact that they are discarded after birth and present large amount of ECM. Decellularization methods can be classified into chemical, physical and enzymatic. All of them are known to cause changes on ECM; thus their association has been commonly used. This study compared different protocols and established a more favorable method for decellularization of canine placentas, aiming at the production of a biomaterial for clinical applications. Initially both placental portions maternal and fetal were subjected to ten different protocols that evaluated variables such as concentration and time of incubation in detergents, different temperatures and the influence of perfusion versus immersion of solutions in the remaining ECM were analysed. The analysis of tissue transparence and absence of cellular nuclei in histological slices stained with HE, led to selection of two protocols (I and II). Besides the before mentioned criteria, both protocols were compared according to the amount of DNA that remained in the ECM decelullarized and the distribution of some ECM proteins. SDS was the most effective detergent for cell removal although it was not enough to complete decellularization. The freezing of placentas led to larger periods of samples incubation in different detergents. Both perfusion and immersion methods were capable of removing cells, although large concentration of cytoskeletal proteins remained entrapped in the matrix. Samples subjected to protocol I (1% SDS, 5 mM EDTA &#43; 50 mM TRIS &#43; 0,5% antibiotic, and 1% Triton X-100) better preserved the structural organization of ECM when compared to those subjected to protocol II (wich differed from the first by the use of 0,05% trypsin instead of 50mM TRIS). However, protocol II optimized cell removal as observed in histological slices and decreased the DNA concentration. Both maternal and fetal portions, subjected to both protocols, retained the laminin, fibronectin and collagen type I proteins. Collagen type III was identified only in fetal portion. In conclusion, protocol II was more effective in the decellularization of canine placentas than protocol I; it removed cellular content and decrease the concentration of remaining DNA in remaining ECM. The ability of ECM decellularized by such method to be applied in tissue engineering strategies still need to be evaluated in vitro and in vivo

A engenharia tecidual visa repor, reparar ou ajudar a regenerar tecidos e órgãos danificados por meio da combinação de biomateriais, biomoléculas e células. Scaffolds de nanofibras biodegradáveis mimetizam a matriz extracelular natural fornecendo uma estrutura ideal para o crescimento celular. Blendas de policaprolactona (PCL) e gelatina são biodegradáveis e proporcionam uma combinação de boas propriedades mecânicas, do PCL, com a hidrofilicidade e caráter que promove a adesão celular, da gelatina. Neste contexto, o objetivo deste trabalho é avaliar a importância das diferentes espessuras de scaffolds eletrofiados em relação às suas propriedades principais. Quatro conjuntos de scaffolds de PCL/gelatina com diferentes espessuras foram produzidos sob as mesmas condições apenas aumentando o tempo de duração do processo de eletrofiação. Os resultados indicam que as espessuras aumentaram proporcionalmente ao tempo de eletrofiação, variando de 100 nm a 300 nm nos períodos de 1 a 3 horas, enquanto a densidade aparente e a porosidade mantiveram-se constantes. As micrografias das membranas revelaram fibras lisas com diâmetros maiores para os scaffolds de menor espessura, e fibras irregulares com diâmetros menores e regiões fundidas ou ligadas para os scaffolds de maior espessura. Além disso, o aumento da espessura melhorou a resistência mecânica e a molhabilidade dos scaffolds. A esterilização por peróxido de hidrogênio não modificou quimicamente a composição das membranas de PCL/gelatina, embora algumas amostras tenham se deformado. As membranas também apresentaram bons resultados de citotoxicidade, melhorando a viabilidade celular, apesar desses valores diminuírem minimamente para os scaffolds de maior espessura, provavelmente devido à maior quantidade de PCL. O teste de adesão não foi conclusivo e deverá ser repetido. / Tissue engineering aims to replace, repair, or helping regenerate damaged tissues and organs through the combination of biomaterials, biomolecules and cells. Biodegradable nanofibrous scaffolds mimic the natural extracellular matrix providing an ideal structure to cellular growth. Blends of polycaprolactone (PCL) and gelatin are biodegradable and provide a combination of good mechanical properties, from PCL, with the hydrophilicity and cell adhesion promoter character, from gelatin. The aim of this work was to evaluate the importance of the thickness of electrospun scaffolds on their key properties. Four sets of PCL/gelatin scaffolds with different thicknesses were produced under the same conditions by simply increasing the time length of electrospinning process. Results indicate that the thickness increases proportionally to the electrospinning time, varying from 100 nm to 300 nm in periods of 1 to 3 hours, while the apparent density and porosity remained constant. Micrographs from the nonwoven mats revealed smooth fibers with larger diameters in the thinner scaffold, and irregular fibers with smaller diameters and molten or bonded regions as the thickness increased. Furthermore, the increase of thickness improved mechanical resistance and wettability of the scaffolds. Plasma sterilization did not modify chemical composition of PCL/gelatin membranes, although some samples have been deformed. Membranes also presented good results for cytotoxicity, improving cell viability, despite these values decreased minimally to the thicker scaffolds, probably due to the higher amount of PCL. Adhesion test was not conclusive and might be repeat.

A promising approach to overcome thrombus and neointima formation on vascular grafts is to create a functional, quiescent monolayer of endothelial cells on the surface of implants. Surface topography of these implants is proven to enhance cell attachment and to reduce the inflammation associated with a smooth surface. Photoembossing is a relatively new, simple, environment-friendly and cost-effective technique to create surface topographies, since there is no etching step or mould needed. In this study, photopolymer films are photoembossed through contact mask photoembossing, while fibres are photoembossed through holographic lithography. Surface relief textures of ridges and grooves with various pitch sizes and heights are successfully obtained through both methods. Furthermore, we introduce this technique to fabricate, for the first time, reproducible surface textures on electrospun fibres. Human umbilical vein endothelial cells (HUVECs) are used in the study. Three different systems are investigated: non-degradable PMMA-TPETA, semi-degradable PLGA-TPETA and fully degradable PLGA-PEGDA-DTT, for different applications and therapeutic requirements. Both non-degradable PMMA-TPETA photopolymer and semi-degradable PLGA-TPETA photopolymer are shown to improve biocompatibility compared to PMMA and PLGA, respectively. Photoembossed films made from these two photopolymers show significantly improved cell attachment and proliferation, IV with a water contact angle around 70º. It is shown that the pitch size of surface topographies affects cell adhesion and migration in the wound healing assay study. Interaction between HUVECs and fibres shows that cells grow from their initial locations at fibre crossings. Focal adhesions are seen to be more aggregated on the surface textured fibres, while those on the glass cover slips are more dispersed near the edge of the cell membrane. The appearance of F-actin in the cytoplasm is also seen to be influenced by the surface topography, where changes in the diameter of the fibre and its surface texture result in F-actin rearrangement. Our study shows that a surface textured, fully degradable, gel-like photopolymer PLGA-PEGDA-DTT has great potential to be further developed for tissue engineering applications.

Cardiovascular disease is the leading cause of death in the United States, accounting for approximately 25% of total deaths. Myocardial infarction (MI) is an extreme case of cardiovascular disease where ischemia leads to irreversible tissue necrosis. As the heart lacks the capacity to endogenously regenerate, the infarcted region is negatively remodeled, reducing cardiac function. Current therapies are not able to regenerate cardiac function post-MI, requiring novel approaches such as tissue engineering. However, there are three major pitfalls that are currently limiting the clinical translation of a tissue engineered cardiac patch: lack of proper vascularization within the tissues; biocompatible material; and lack of electrical integration between engineered tissue and host. The research within this dissertation aimed to engineer solutions to overcome these three pitfalls. Plants and animals exploit fundamentally different approaches to transporting fluids, yet there are surprising structural similarities. To take advantage of these similarities, we looked across different kingdoms and investigated whether plants and their innate vasculature could serve as perfusable scaffolds for tissue engineering. Standard perfusion decellularization techniques were adapted and applied to spinach leaves, which were found to be fully devoid of DNA following processing. Leaf vasculature remained patent post-decellularization and supported transport of various sized microparticles. Human cells successfully seeded onto and inside the plant scaffolds. Decellularized leaves were found to be nearly void of any cytotoxic affects. Leaf biocompatibility was then investigated in vivo through subcutaneous implantation in a rat model. Leaf scaffolds were found to be biocompatible after 4 weeks of implantation. Furthermore, leaves that were pre-functionalized with an RGD-dopamine peptide were fully integrated into the host tissue within one week. This shows the leaf scaffold’s potential to be an immuno-modulatory material, depending upon the intended application. Electrically conducting biofibers were engineered through the combination of fibrin microthreads and engineered conductive HEK293 cells. Biofibers could act as a modular platform to allow for electrical integration between the host tissue and any engineered cardiac patch. Biofibers directionally carried electrical current and were found capable of bridging electrical signal between two separate clusters of cardiomyocytes. In vivo investigation bridging a biofiber from the left atria to the left ventricle was accomplished in a rat model. Electrical maps demonstrated a visible accessory pathway that created a feedback electrical signal from the ventricle to the atria through the implanted biofiber. These results demonstrate electrical integration in vivo between host myocardium and the engineered biofiber.