Anthropomorphic phantoms are commonly used for testing radiation fields without the need to expose human subjects. One of the most widely known is RANDO phantom. This phantom is used primarily for medical X-ray applications, but a similar design known as "MATROSHKA" is now being used for space research and exposed to heavy ion irradiations from the Galactic environment. Since the radiation field in the phantom should respond in a similar manner to how it would act in human tissues and organs under an irradiation, the tissue substitute chosen for soft tissue and the level of complexity of the entire phantom are crucial issues. The phantoms, and the materials used to create them, were developed mainly for photon irradiations and have not been heavily tested under the conditions of heavy ion exposures found in the space environment or external radiotherapy. The Particle and Heavy-Ion Transport code System (PHITS) was used to test the phantoms and their materials for their potential as human surrogates for heavy ion irradiation. Stopping powers and depth-dose distributions of heavy charged particles (HCPs) important to space research and medical applications were first used in the simulations to test the suitability of current soft tissue substitutes. A detailed computational anthropomorphic phantom was then developed where tissue substitutes and ICRU-44 tissue could be interchanged to verify the validation of the soft tissue substitutes and and determine the required level of complexity of the entire phantom needed to achieve a specified precision as a replacement of the human body. The materials tested were common soft tissue substitutes in use and the materials which had a potential for the soft tissue substitute. Ceric sulfate dosimeter solution was closest to ICRU-44 tissue; however, it was not appropriate as the phantom material because it was a solution. A150 plastic, ED4C (fhw), Nylon (Du Pont Elvamide 8062), RM/SR4, Temex, and RW-2 were within 1% of the mean normalized difference of mass stopping powers (or stopping powers for RW-2) when compared to the ICRU-44 tissue, and their depth-dose distributions were close; therefore, they were the most suitable among the remaining solid materials. Overall, the soft tissue substitutes which were within 1% of ICRU-44 tissue in terms of stopping power produced reasonable results with respect to organ dose in the developed phantom. RM/SR4 is the best anthropomorphic phantom soft tissue substitute because it has similar interaction properties and identical density with ICRU-44 tissue and it is a rigid solid polymer giving practical advantages in manufacture of real phantoms.

Osteochondral defects comprise cartilage and bone tissue in the joint region and create challenges for orthopedic surgery, also because intrinsic regeneration capacities of the articular cartilage are limited. Furthermore, tissue layer-specific characteristics regarding cell types, mechanical properties and biochemical composition need to be considered. Research questions: In this work, concepts were developed which allow mimicking of osteochondral interfacial layers in a patient-individual and zonally specified manner by 3D extrusion (bio)printing. This feature of patient specificity was proven on different levels within this project: Besides the option for application of patient-own, expanded stem cells or chondrocytes within a scaffold to support regeneration and neo-tissue formation, a workflow was implemented which enables the consideration of magnetic resonance imaging (MRI) data and zonal geometry of the defect. With the materials suitable to achieve this design and a bioprinting-compatible process, the impact of such a system on embedded cells was investigated. A zonally structured, partly mineralized construct was evaluated regarding its capability to allow or support chondrogenesis of primary human chondrocytes (hChon). Furthermore, a strategy based on core-shell bioprinting technology was developed which allows simultaneous embedding of different cell types in a zonally defined distribution with a targeted effect by incorporated growth factors while reducing the off-target effects that would be expected when applied homogeneously via the surrounding medium. In addition, hybrid multi-material scaffolds were developed to adjust the stiffness of these systems. Materials and methods: To define design and patient-specific requirements for an osteochondral implant, an anonymized MRI dataset of a patient with osteochondritis dissecans (OCD) was used. The main constituent of the developed fabrication system was a bioink based on 3% alginate and 9% methylcellulose (algMC) with hChon. Laponite was added to alg-MC-based inks in order to control the release of differentiation factors for a sustained delivery in multi-zonal osteochondral constructs. A printable calcium phosphate cement (CPC) was used as a mineral phase. For the bioprinting process, multi-channel extrusion was applied for an alternating printing of hChon-laden algMC and CPC in order to mimic a zone of mineralized cartilage. Cell fate was investigated on biochemical and gene expression level. A coaxial extrusion module was applied for the co-extrusion of a bioink (shell) – algMC or plasma-functionalized algMC loaded with hChon or human pre-osteoblasts (hOB), respectively – and a biomaterial ink (core) doped with the corresponding growth factors TGF-β3 or BMP-2 as central target-specific factor depot. By melt electrowriting technology (MEW), additional scaffolds from polycaprolactone (PCL) microfibers with a freely adjustable fiber structure were generated. To trigger the mechanical stiffness of cell-laden hydrogels, these scaffolds were manually added to the bioprinting process as an extra support. Results: Suggested strategies of 3D extrusion (bio)printing for clinically relevant dimensions (Publication I)were successfully applied on algMC-based inks, bioinks and CPC to generate multi-material cell-laden constructs of an individual, patient-specific shape. With the use of flexible and reversible software solutions, MRI data from an OCD patient were utilized for the design and later fabrication of a bi-zonal implant (Publication II). The resulting implant showed a suitable geometry fitting into a model of the lesioned femoral condyles fabricated by stereolithography. For surgical fixation of such a potential implant, an individual implantation adapter was developed. The same materials processable via multi-channel printing were compatible with bioprinting of hChon isolated from the femoral head of human hip arthroplasty patients. The majority of cells survived the printing process and cultivation conditions in monophasic scaffolds consisting of cell-laden algMC, and in biphasic scaffolds with a zonally separated or interwoven mineral zone of calcium phosphate cement. Cells in both setups, representing plain articular cartilage and calcified cartilage, were able to re-differentiate and demonstrated the characteristic ECM marker production and gene expression. The calcium-deficient CPC led to a decrease of calcium ions and an initial increase of phosphate ions in the surrounding medium. In the presence of the CPC phase, chondrogenesis was enhanced (Publication III). The core-shell bioprinting concept allowed the spatially defined differentiation of cells (hChon or hOB), encapsulated in a bioink extruded as shell compartment, adjacent to a respective factor-loaded core depot with specific differentiation factors. The biomaterial inks for the core depot were successfully adjusted regarding viscosity and release kinetics by addition of nanoclay (Laponite) nanoparticles. Optical coherence tomography (OCT) was introduced as a tool to monitor the coaxial strand pattern and the location of embedded cells in a contactless manner. The applied inks allowed adjustment of release properties of components such as growth factors BMP-2 and TGF-β3. In hChon, characteristic genes such as collagen 2 or aggrecan were upregulated, while hOB were able to express the typical genes ALP, BGLAP and IBSP. Although both incorporated differentiation factors also demonstrated enhancing effects on both compartments, respectively, the induced adverse effects of hypertrophy in the cartilage zone and collagen 2 expression in the bone zone were successfully prevented. This was done by applying the factors with a sustained release via a Laponite-supported ink as the core depots, instead of homogeneously supplementing the surrounding cell culture medium (Publication IV). By adding PCL microfiber mesh scaffolds, fabricated by MEW, with a decreasing fiber density from 1000 to 250 µm, the Young’s modulus of the algMC scaffolds increased from 10 kPa to more than 50 kPa. The resulting hybrid scaffolds were proven cytocompatible; bioprinted hChon reacted to this hybrid algMC structure with a PCL density of 750 µm with an improved release of sulphated glycosaminoglycans (Publication V). Conclusions: A fully integrated approach for a multiphasic implant design, embedding of primary cells and simultaneous application of respective growth factors was realized by 3D extrusion (bio)printing. Concepts for bioprinting of mineralized cartilage based on algMC and CPC and for local factor delivery in osteochondral tissue substitutes by core-shell bioprinting were developed. The presented approaches allow an adjustable zonal design and full control over spatial differentiation and fate of bioprinted cells. The versatility of this modular system allows addition of further features as demonstrated for the combination with PCL microfiber scaffolds to adjust mechanical properties of the cartilage zone. Another option can be the mechanical stimulation of magnetically deformable algMC-magnetite scaffolds. These valuable insights for the field will serve as basis for further applications in vitro and in vivo. They might open up new research directions with a potential translation to other material combinations and other tissue defect types.:Table of Contents List of abbreviations List of figures Legal note 1. Introduction 1.1 The osteochondral interface – function, anatomy and histology 1.2 Pathology of cartilage and osteochondral tissue 1.3 State of the art: treatment of cartilage defects and osteochondral defects 1.4 Tissue engineering for osteochondral regeneration 1.5 Biomedical additive manufacturing and bioprinting 1.6 Hydrogels for bioprinting 1.7 Multi-component and multiphasic strategies to add specific cues and features to bioprinted tissue models 1.8 Additive Manufacturing of patient-specific bone and cartilage substitutes 2. Aims of the thesis List of publications included in the thesis 3. Strategies for biofabrication of volumetric constructs with an individual shape (Publication I) Publication I: Review article 4. Workflow for an MRI-guided, bi-zonal implant design (Publication II) 41 Publication II: Article Publication II: Published supporting information 5. Chondrogenesis in 3D bioprinted constructs and its compatibility with a mineral phase (Publication III) Publication III: Article Publication III: Published supporting information 6. Concept for a zonally defined factor delivery (Publication IV) Publication IV: Article Publication IV: Published supporting information 7. Hybrid bioscaffolds for tailoring mechanical properties of cartilage tissue substitutes (Publication V) Publication V: Article 8. Discussion and outlook References SUMMARY ZUSAMMENFASSUNG Acknowledgements List of other publications (co-)authored by the candidate Scientific congress contributions during PhD phase Journal ranking in Journal Citations Report Appendix I – Erklärungen zur Eröffnung des Promotionsverfahrens Appendix 2 – Erklärung zur Einhaltung gesetzlicher Bestimmungen / Osteochondrale Defekte umfassen Knochen- und Knorpelgewebe innerhalb des betroffenen Gelenks und stellen die klinische Orthopädie vor Herausforderungen dar, auch da die intrinsische Regenerationsfähigkeit des Gelenkknorpels stark limitiert ist. Zudem sind in den zu unterscheidenden Gewebeschichten spezifische Charakteristika wie unterschiedliche Zelltypen, mechanische Eigenschaften und die biochemische Zusammensetzung zu berücksichtigen. Fragestellungen: In der vorliegenden Arbeit wurden Konzepte entwickelt, mit dem sich per 3D-Extrusions(bio)druck Gewebeschichten dieser osteochondralen Grenzschicht zonenspezifisch und patientenindividuell nachbilden lassen. Diese patientenindividuellen Merkmale wurden innerhalb des Projektes auf mehreren Ebenen nachgewiesen: Zum einen können patienteneigene Stammzellen oder Chondrozyten nach Vermehrung im Labor innerhalb einer Gerüststruktur (“Scaffold”) zur Unterstützung der Regeneration und Gewebeneubildung angewandt werden. Zum anderen wurde ein Workflow vorgestellt, der die Berücksichtigung einer individuellen, per Magnetresonanztomographie (MRT) detektierten, schichtweisen Geometrie einer Läsion erlaubt. Mit Hilfe von Materialien, die diese Formgebung ermöglichen, wurde in einem Biodruck-kompatiblen Prozess der Einfluss eines solchen Systems auf eingebettete Zellen untersucht: Ein zonal aufgebautes, teilweise mineralisiertes Konstrukt wurde hinsichtlich dessen Eignung, Chondrogenese humaner Knorpelzellen (hChon) zu ermöglichen oder zu unterstützen, evaluiert. Zudem wurde eine auf der Kern-Mantel-Biodrucktechnologie basierende Strategie entwickelt, die das Einbetten unterschiedlicher Zelltypen mit zonal definierter Verteilung kombiniert mit einem gezielten Effekt durch inkorporierte Wachstumsfaktoren. Hierbei sollten unerwünschte Nebeneffekte der im Kern dargebrachten Faktoren auf die jeweils andere Zellsorte, die man bei homogener Faktorengabe über das umgebende Medium erwarten würde, reduziert werden. Weiterhin sollte mittels hybrider Multi-Material-Scaffolds die Steifigkeit des Systems angepasst werden. Material und Methoden: Um ein Design und patientenindividuelle Anforderungen für ein osteochondrales Implantat zu definieren, wurde ein anonymisierter MRT-Datensatz eines Osteochondrosis dissecans(OCD)-Patienten genutzt. Hauptbestandteil des entwickelten Fabrikationssystems war eine Biotinte aus 3% Alginat und 9% Methylcellulose (algMC) mit hChon. Laponit wurde zu den auf algMC basierenden Tinten hinzugefügt, um die Freisetzung von Differenzierungsfaktoren zu kontrollieren und damit eine verzögerte Gabe in mehrschichtigen osteochondralen Konstrukten zu ermöglichen. Ein druckbarer Kalziumphosphatzement (CPC) wurde als Mineralphase genutzt. Im Biodruckprozess wurde der Mehrkanaldruck angewandt, um durch alternierende Extrusion von hChon-beladenem algMC und CPC die mineralisierte Knorpelschicht nachzubilden. Die Zellentwicklung wurde auf biochemischer Ebene und hinsichtlich der exprimierten Gene untersucht. Ein koaxiales Extrusionsmodul wurde zur Ko-Extrusion einer Biotinte (Mantel), bestehend aus algMC beladen mit hChon oder Plasma-funktionalisierter algMC beladen mit humanen Prä-Osteoblasten (hOB), und einer korrespondierenden faktorenbeladenen Biomaterialtinte (Kern) genutzt. Dieses zielspezifische Faktorendepot enthielt jeweils TGF-β3 oder BMP-2. Durch die Technik des Melt Electrowritings (MEW) wurden zusätzliche Scaffolds aus Polycaprolacton(PCL)-Mikrofasern mit einer justierbaren Faserstruktur generiert. Um die Steifigkeit von zellbeladenen Hydrogelen anzupassen, wurden diese Scaffolds als mechanischer Support manuell während des Biodruckprozesses eingebracht. Ergebnisse: Die zugrundeliegenden Strategien des 3D-Extrusions(bio)drucks in klinisch relevanten Dimensionen (Publikation I) wurden an algMC-basierten Tinten, Biotinten und CPC erfolgreich angewandt, um zellbeladene Konstrukte patientenindividueller Form aus mehreren Materialien zu generieren. Durch den Einsatz flexibler und reversibler Software-Lösungen, wurden MRT-Daten eines Patienten mit einem osteochondralen Defekt verwendet, um ein zweischichtiges Implantatdesign zu entwerfen und zu fertigen (Publikation II). Dieses Implantat wies eine adäquate Passgenauigkeit in einem Modell der Läsion in den Femurkondylen, hergestellt per Stereolithografie, auf. Zur chirurgischen Fixierung eines solchen potenziellen Implantats wurde ein individueller Adapter für einen chirurgischen Stößel entwickelt. Das gleiche Materialsystem, prozessierbar mittels Mehrkanaldrucks, erwies sich als kompatibel zum Biodruck von hChon, isoliert aus dem Femurkopf von Hüft-Totalendoprothese-Patienten. Die meisten der Zellen überlebten den Druckprozess und die Kultivierungsbedingungen in monophasigen Scaffolds bestehend aus zellbeladener algMC-Biotinte, sowie in biphasigen Scaffolds mit einer in einer getrennten Schicht verlaufenden oder verwobenen mineralisierten Zone aus CPC. Zellen waren in beiden Ansätzen, als monophasiger oberflächlichen Gelenkknorpel, sowie als kalzifizierte Knorpelschicht, in der Lage, sich zu redifferenzieren; sie zeigten die Expression charakteristischer Matrix-Komponenten und -Gene. Der Kalzium-defizitäre CPC führte zu einer Verminderung der Kalziumionenkonzentration und zu einem initialen Anstieg der Phosphationen im umgebenden Medium. In Gegenwart der CPC-Phase war die Chondrogenese verstärkt (Publikation III). Das Konzept des Kern-Mantel-Biodrucks ermöglichte die örtlich aufgelöste Differenzierung von Zellen (hChon oder hOB), eingebettet in eine Biotinte extrudiert als Mantel-Kompartment, in unmittelbarer Nähe zu einem entsprechenden Faktor-beladenen Depot mit spezifischen Differenzierungsfaktoren. Die Biomaterialtinten für das Kern-Depot wurden durch die Zugabe von Nanoclay(Laponit)-Nanopartikeln hinsichtlich Viskosität und Freisetzungskinetik erfolgreich angepasst. Optische Kohärenztomographie (OCT) wurde als eine zerstörungsfreie Methode zur Beobachtung des koaxialen Strangmusters und der Zellverteilung eingeführt. Die genutzten Tinten erlaubten die Adaption der Freisetzungskurven unterschiedlicher Moleküle wie der Wachstumsfaktoren BMP-2 und TGF-β3. In hChon war die Expression charakteristischer Gene wie Kollagen 2 oder Aggrecan verstärkt, während hOB die für die osteogene Differenzierung typischen Markergene ALP, BGLAP und IBSP exprimierten. Obwohl beide inkorporierten Faktoren auch verstärkende Effekte auf jeweils beide Kompartimente zeigten, konnte der induzierte unerwünschte Effekt der Hypertrophie innerhalb der Knorpelzone sowie die unerwünschte Kollagen Typ 2-Expression innerhalb der Knochenzone erfolgreich verhindert werden. Dies geschah, indem die Faktoren statt homogen über das umgebende Zellkulturmedium mittels Laponit-Tinte und daher freisetzungsverzögernd über die Kern-Depots dargereicht wurden (Publikation IV). Mittels der PCL-Mikrofaser-Gitter-Scaffolds, hergestellt per MEW, mit enger werdenden Fasernetzdichten von 1000 bis 250 µm konnte der E-Modul der algMC-Scaffolds von 10 kPa auf über 50 kPa erhöht werden. Die Zytokompatibilität der hybriden Scaffolds wurden nachgewiesen; auf die Struktur in hybriden algMC-Scaffolds mit einer PCL-Faserdiche von 750 µm reagierten biogedruckte hChon mit einer erhöhten Freisetzung von sulfatierten Glykosaminoglykanen (Publikation V). Schlussfolgerungen: Ein integrierter Ansatz für ein mehrphasiges Implantatdesign, das Einbetten von primären Zellen und die gleichzeitige Anwendung der entsprechenden Wachstumsfaktoren wurde mittels 3D-Extrusions(bio)druck realisiert. Konzepte zum Biodruck von mineralisiertem Knorpel basierend auf algMC und CPC und zur lokalen Faktorengabe in osteochondralen Gewebeersatzstrukturen per Kern-Mantel-Druck wurden entwickelt. Die vorgestellten Ansätze erlauben ein vielseitig adaptierbares, zonales Design, die volle Kontrolle über die örtliche Differenzierung sowie die Reifung der biogedruckten Zellen. Die Vielseitigkeit des modularen Systems ermöglicht zudem das Hinzufügen weiterer Merkmale, was anhand des Einbringens von PCL-Mikrofaser-Scaffolds zur Justierung der mechanischen Eigenschaften der Knorpelzone demonstriert wurde. Eine weitere Option stellt die mechanische Stimulation magnetisch verformbarer algMC-Magnetit-Scaffolds dar. Die wertvollen Erkenntnisse werden als Basis für weitere Anwendungen in vitro sowie in vivo dienen können. All dies kann neue Möglichkeiten und Forschungsrichtungen eröffnen und ist in vielerlei Hinsicht übertragbar auf weitere Materialkombinationen, sowie verschiedene Defekt- und Gewebearten.:Table of Contents List of abbreviations List of figures Legal note 1. Introduction 1.1 The osteochondral interface – function, anatomy and histology 1.2 Pathology of cartilage and osteochondral tissue 1.3 State of the art: treatment of cartilage defects and osteochondral defects 1.4 Tissue engineering for osteochondral regeneration 1.5 Biomedical additive manufacturing and bioprinting 1.6 Hydrogels for bioprinting 1.7 Multi-component and multiphasic strategies to add specific cues and features to bioprinted tissue models 1.8 Additive Manufacturing of patient-specific bone and cartilage substitutes 2. Aims of the thesis List of publications included in the thesis 3. Strategies for biofabrication of volumetric constructs with an individual shape (Publication I) Publication I: Review article 4. Workflow for an MRI-guided, bi-zonal implant design (Publication II) 41 Publication II: Article Publication II: Published supporting information 5. Chondrogenesis in 3D bioprinted constructs and its compatibility with a mineral phase (Publication III) Publication III: Article Publication III: Published supporting information 6. Concept for a zonally defined factor delivery (Publication IV) Publication IV: Article Publication IV: Published supporting information 7. Hybrid bioscaffolds for tailoring mechanical properties of cartilage tissue substitutes (Publication V) Publication V: Article 8. Discussion and outlook References SUMMARY ZUSAMMENFASSUNG Acknowledgements List of other publications (co-)authored by the candidate Scientific congress contributions during PhD phase Journal ranking in Journal Citations Report Appendix I – Erklärungen zur Eröffnung des Promotionsverfahrens Appendix 2 – Erklärung zur Einhaltung gesetzlicher Bestimmungen

Made available in DSpace on 2015-03-26T13:47:10Z (GMT). No. of bitstreams: 1 texto completo.pdf: 2556452 bytes, checksum: d5563353a41b374f1b598abd2f574cf3 (MD5) Previous issue date: 2012-09-04 / Corneal conditions are among the main causes of blindness in both humans and animals. Many surgical techniques are described to recover the cornea s physical structure and refractive state; among them is the use of different types of biomaterials. Due to the cornea s transparency, viscoelasticity and lack of vessels, biomaterials to be applied on them must undergo a careful selection. Bacterial cellulose (BC) and polycaprolactone (PCL) are two well known biomaterials, studied extensively in separate. The purpose of the present study was to produce and characterize membranes made of BC and PCL to be tested as corneal tissue substitutes in rabbit s cornea. Membranes of BC were produced through static culture of the bacteria Gluconacetobacter xylinus. Membranes were then washed, dried and swelled in acetone for 48 hours, followed by immersion in 25ml of 3% PCL solution for 72 hours. Next, membranes of the BC/PCL composite underwent several washes in acetone and were then dried to be characterized by scanning electron microscopy (SEM), X-ray diffraction, thermal and mechanical trials and luminous transmittance. In vivo study was conducted with the BC/PCL composite and with pure BC to evaluate their interactions with corneal tissue. At first, membranes were cut in 8mm diameter to be then applied in corneal tisse. Thirty six female rabbits were separated in three groups of 12 animals each. An ulcer of 5mm in diameter and 0.2mm in depth was created on the right cornea of all rabbits. From the base of the ulcer, the stroma was separated creating an interlamellar pocket, and each group of 12 animals received 8mm diameter BC/PCL composite membrane and the other group received BC membranes on the same size, both adjusted in the pocket. The last group did not receive any membrane, used as the control. Animals were followed for 45 days, where clinical evaluations for signs of pain and inflammation were conducted. Three animals of each group were euthanized on days 3, 7, 21 and 45 after surgery to collect samples for histology (HE and Picrosirius) and SEM. BC/PCL presented 84% of light transmission while pure BC presented 45%. Lower tension resistance and Young s module was observed on the composite compared to pure BC, while the composite presented greater elongation at rupture what demonstrates a greater viscoelasticity of this material. Eyes that received membranes presented moderate inflammation until 30 days after surgery, decreasing progressively until 45 days. Histology revealed the presence of lymphohistiocytic inflammatory infiltrate at 21 and 45 days in implanted groups, at a moderate intensity, as well as giant cells, suggesting chronic inflammatory and foreign body reactions. Fibroplasia was observed around the membranes, a positive sign of biocompatibility. Epithelization was complete only in control group, what shows that membranes surfaces did not allow epithelium growth and maintenance. Picrosirius staining showed a greater proportion of type III collagen at the end of 45 days in both implanted groups, suggesting that the repair process around the membranes was incomplete. SEM showed extracellular matrix and cells adhered to implant surfaces, suggesting integration at 45 days. Both membranes made of BC/PCL composite and BC alone stimulated chronic inflammatory and foreign body reactions and did not allow complete epithelization on their surfaces. Based on these conclusions, we believe that implants made of BC/PCL and BC using the here presented process are not indicated as a substitute for corneal tissue in rabbits. / As afecções corneais representam uma das principais causas de cegueira tanto em humanos como em animais. São descritas várias técnicas cirúrgicas para recuperar a estrutura física e o estado refrativo da córnea, dentre essas, encontra-se a aplicação de diferentes tipos de biomateriais. Devido à transparência, à falta de vasos e à viscoelasticidade que a córnea apresenta, os biomateriais para nela serem aplicados devem passar por criteriosa seleção. A celulose bacteriana (CB) e a policaprolactona (PCL) são dois consagrados biomateriais, bastante estudados isoladamente. Foi proposto, neste estudo, a produção e a caracterização de membranas à base de CB e de PCL para serem testadas como substituto tecidual em córneas de coelhos. Membranas de CB foram produzidas por meio do cultivo estático da bactéria Gluconacetobacter xylinus. Após serem lavadas e secas, as membranas foram intumescidas em acetona por 48 horas e então imersas em 25 ml de solução a 3% de PCL diluída em acetona durante 72 horas. Na sequência, as membranas do compósito CB/PCL passaram por repetidas lavagens em acetona, e foram secas para serem caracterizadas por microscopia eletrônica de varredura (MEV), difração de raio-X, ensaios térmico e mecânico e transmissão óptica. Foi realizado o estudo in vivo com membranas do compósito CB/PCL e da CB pura para avaliar suas interações com o tecido corneal. Inicialmente elas foram cortadas em 8 mm de diâmetro para então serem implantadas nas córneas dos animais. Foram utilizadas 36 coelhas divididas em três grupos de 12 animais. Foi criada uma úlcera de 5 mm de diâmetro e 0,2 mm de profundidade na córnea direita de todos os animais. A partir da base da úlcera o estroma foi separado, criando-se um bolso interlamelar, sendo que um grupo recebeu membranas do compósito CB/PCL e outro grupo recebeu membranas da CB, que foram ajustadas dentro do bolso. O último grupo não recebeu tratamento sendo utilizado como controle. Os animais foram acompanhados por até 45 dias, sendo feitas avaliações clínicas para os sinais de dor e inflamação. Três animais de cada grupo foram eutanasiados aos três, sete, 21 e 45 dias após a cirurgia, para coleta de amostras para análises histológicas (HE e Picrosirius) e MEV. O compósito CB/PCL apresentou 84% de transmissão de luz, enquanto que a CB pura apresentou 65%. Houve redução da resistência à tração e do módulo de Young do compósito CB/PCL em relação à CB pura. Em contrapartida, o compósito apresentou aumento na ruptura ao alongamento, demonstrando incremento na elasticidade do material. Os olhos que receberam implantes apresentaram inflamação moderada até os 30 dias de pós-operatório, reduzindo progressivamente até os 45 dias da cirurgia. A análise histológica demonstrou, aos 21 e aos 45 dias, presença de infiltrado inflamatório linfo-histiocitário em intensidade moderada e células gigantes nos animais implantados, sugerindo resposta inflamatória persistente e do tipo corpo estranho. Também foi observada fibroplasia em torno das membranas, sendo este um sinal positivo de biocompatibilidade. A epitelização foi completa somente no grupo controle, demonstrando que as superfícies das membranas não permitiram o crescimento e manutenção do epitélio. Pela coloração de Picrosirius, evidenciou-se maior proporção do colágeno tipo III ao final dos 45 dias nos grupos com implantes, sugerindo que o processo de reparação em torno das membranas estava incompleto. A MEV demonstrou matriz extracelular e células aderidas às superfícies dos implantes, sinalizando integração no período de 45 dias. As membranas tanto do compósito CB/PCL como da CB incitaram resposta inflamatória crônica e de corpo estranho, e não permitiram a completa epitelização sobre suas superfícies. Baseado nisso, acredita-se que os implantes CB/PCL e CB, da maneira como foram desenvolvidos, não devem ser indicados para substituir o tecido corneal de coelhos.