Decellularization and Delipidation Protocols of Bovine Bone and Pericardium for Bone Grafting and Guided Bone Regeneration Procedures

The combination of bone grafting materials with guided bone regeneration (GBR) membranes seems to provide promising results to restore bone defects in dental clinical practice. In the first part of this work, a novel protocol for decellularization and delipidation of bovine bone, based on multiple steps of thermal shock, washes with detergent and dehydration with alcohol, is described. This protocol is more effective in removal of cellular materials, and shows superior biocompatibility compared to other three methods tested in this study. Furthermore, histological and morphological analyses confirm the maintenance of an intact bone extracellular matrix (ECM). In vitro and in vivo experiments evidence osteoinductive and osteoconductive properties of the produced scaffold, respectively. In the second part of this study, two methods of bovine pericardium decellularization are compared. The osmotic shock-based protocol gives better results in terms of removal of cell components, biocompatibility, maintenance of native ECM structure, and host tissue reaction, in respect to the freeze/thaw method. Overall, the results of this study demonstrate the characterization of a novel protocol for the decellularization of bovine bone to be used as bone graft, and the acquisition of a method to produce a pericardium membrane suitable for GBR applications

Guided Tissue Regeneration in Heart Valve Replacement: From Preclinical Research to First-in-Human Trials

Heart valve tissue-guided regeneration aims to offer a functional and viable alternative to current prosthetic replacements. Not requiring previous cell seeding and conditioning in bioreactors, such exceptional tissue engineering approach is a very fascinating translational regenerative strategy. After in vivo implantation, decellularized heart valve scaffolds drive their same repopulation by recipient’s cells for a prospective autologous-like tissue reconstruction, remodeling, and adaptation to the somatic growth of the patient. With such a viability, tissue-guided regenerated conduits can be delivered as off-the-shelf biodevices and possess all the potentialities for a long-lasting resolution of the dramatic inconvenience of heart valve diseases, both in children and in the elderly. A review on preclinical and clinical investigations of this therapeutic concept is provided with evaluation of the issues still to be well deliberated for an effective and safe in-human application

Biological Scaffolds for Congenital Heart Disease

Congenital heart disease (CHD) is the most predominant birth defect and can require severalinvasive surgeries throughout childhood. The absence of materials with growth and remodellingpotential is a limitation of currently used prosthetics in cardiovascular surgery, as well as theirsusceptibility to calcification. The field of tissue engineering has emerged as a regenerative medicineapproach aiming to develop durable scaffolds possessing the ability to grow and remodel uponimplantation into the defective hearts of babies and children with CHD. Though tissue engineeringhas produced several synthetic scaffolds, most of them failed to be successfully translated in this lifeendangeringclinical scenario, and currently, biological scaffolds are the most extensively used. Thisreview aims to thoroughly summarise the existing biological scaffolds for the treatment of paediatricCHD, categorised as homografts and xenografts, and present the preclinical and clinical studies.Fixation as well as techniques of decellularisation will be reported, highlighting the importanceof these approaches for the successful implantation of biological scaffolds that avoid prostheticrejection. Additionally, cardiac scaffolds for paediatric CHD can be implanted as acellular prostheses,or recellularised before implantation, and cellularisation techniques will be extensively discussed

New strategies for the decellularization of biological tissues

O início do século 21 tem sido marcado pelo aumento das doenças crónicas. Este desenvolvimento resultou num crescimento do interesse na criação de novas terapias, com foco na recuperação de tecidos através da transplantação do tecido danificado por matrizes “inteligentes” desenvolvidas recorrendo ao uso da Engenharia Biomédica. A descelularização, um processo que visa a remoção de material celular imunogénico de um tecido ou órgão, tem-se tornado num meio atraente para o desenvolvimento de novas matrizes funcionais e bioativas. A presente tese teve como objetivo explorar novas metodologias para a descelularização de tecidos biológicos. Para este propósito, é apresentada uma revisão da literatura relevante e um estudo que investiga o potencial de três diferentes protocolos para a descelularização de osso trabecular porcino usando o fosfato de tri-n-butilo (TnBP), dióxido de carbono supercrítico (scCO2), e uma combinação de ambos. O uso do TnBP como um agente de descelularização, ao invés do uso de produtos químicos mais prejudiciais como os detergentes, pode levar a uma maior preservação da matriz extracelular (ECM), tal como a propriedades bioquímicas e mecânicas mais desejáveis para a matriz resultante. O uso do scCO2 pode, também, resultar num processo de descelularização mais rápido, levando não só a uma redução do tempo de exposição dos tecidos a produtos químicos potencialmente prejudiciais, mas também a uma redução do preço financeiro deste processo. No total foram implementados e examinados cinco protocolos diferentes: 1% (v/v) TnBP durante 48 horas, scCO2 durante 1 hora e 3 horas, e scCO2 com 0.1% (p/v) TnBP com durante 1 hora e 3 horas. Devido à natureza inovadora deste projeto, usaram-se variáveis temporais para estudar qualquer efeito prejudicial devido ao efeito da exposição prolongada ao scCO2. Os resultados obtidos revelaram que tanto o TnBP como o scCO2 conseguiram diminuir a quantidade de DNA presente nas amostras, mas esta diminuição foi maior nos protocolos que onde o TnBP foi usado. A análise às propriedades mecânicas dos tecidos sujeitos a TnBP revelaram um aumento da força máxima e da tensão de limite elástico, o que poderá significar que ocorreu crosslinking das fibras de colagénio. Já o uso do scCO2 resultou na desidratação das amostras, aumentado os valores para o módulo de Young e força máxima. O protocolo de combinação scCO2-TnBP causou uma diminuição para metade da quantidade de DNA presente nas amostras tratadas em comparação a não-tratadas, demonstrado o potencial desta metodologia inovadora e abrindo novas possibilidade para otimizações futuras.The beginning of the 21st century has been marked by the rise of chronic diseases. This development has led to increased interest in the development of new therapies that focus on restoring normal tissue function through transplantation of injured tissue with biomedically engineered smart matrices. Decellularization, a process that focuses on the removal of immunogenic cellular material from a tissue or organ, has become an appealing methodology for the creation of functional and bioactive scaffolds. The present thesis focused on the creation of new methodologies for the decellularization of biological tissues. For this purpose, the author reviewed current decellularization literature and put forward a study that investigated the potential of three different decellularization protocols for porcine trabecular bone tissue using Tri(n-butyl) phosphate (TnBP), supercritical carbon dioxide (scCO2), and a combination of both. The use of TnBP as a decellularization agent, instead of harsh chemicals such as detergents, could lead to better preservation of the extracellular matrix (ECM), and better biochemical and mechanical properties to the resulting scaffold. As well, the use of supercritical fluids could lead to faster decellularization times, not only reducing the time tissues are exposed to potentially harmful agents, but also reducing the financial cost of the process. In total, five different protocols were implemented and examined: 1% (v/v) TnBP treatment for 48 hours, scCO2 treatment for 1 hour and 3 hours, and scCO2 treatment with 0.1% (w/v) TnBP for 1 hour and 3 hours. Due to the innovative nature of this work, time variants to protocols were implemented to investigate any possible harmful effects caused by prolonged exposure to scCO2 treatment. Results revealed that both TnBP and scCO2 led to the removal of DNA content, but this effect was more pronounced in treatments that used TnBP. Mechanical analysis of TnBP-treated samples revealed a higher ultimate strength and yield strain, suggesting some degree of crosslinking of collagen fibers occurred. Meanwhile, the use of scCO2 led to dehydration of samples, increasing values for Young’s Modulus and ultimate strength. The combined protocol of scCO2-TnBP led to a decrease in DNA content to about half of that measured for untreated samples, demonstrating the potential of this methodology and opening new possibilities for future optimizations that could achieve required decellularization levels

Hemocompatibility tuning of an innovative glutaraldehyde-free preparation strategy using riboflavin/UV crosslinking and electron irradiation of bovine pericardium for cardiac substitutes

Hemocompatibility tuning was adopted to explore and refine an innovative, GA-free preparation strategy combining decellularization, riboflavin/UV crosslinking, and low-energy electron irradiation (SULEEI) procedure. A SULEEI-protocol was established to avoid GA-dependent deterioration that results in insufficient long-term aortic valve bioprosthesis durability. Final SULEEI-pericardium, intermediate steps and GA-fixed reference pericardium were exposed in vitro to fresh human whole blood to elucidate effects of preparation parameters on coagulation and inflammation activation and tissue histology. The riboflavin/UV crosslinking step showed to be less efficient in inactivating extracellular matrix (ECM) protein activity than the GA fixation, leading to tissue-factor mediated blood clotting. Intensifying the riboflavin/UV crosslinking with elevated riboflavin concentration and dextran caused an enhanced activation of the complement system. Yet activation processes induced by the previous protocol steps were quenched with the final electron beam treatment step. An optimized SULEEI protocol was developed using an intense and extended, trypsin-containing decellularization step to inactivate tissue factor and a dextran-free, low riboflavin, high UV crosslinking step. The innovative and improved GA-free SULEEI-preparation protocol results in low coagulant and low inflammatory bovine pericardium for surgical application

Supercritical Carbon Dioxide Facilitated Collagen Scaffold Production for Tissue Engineering

The rise of tissue engineering and regenerative medicine (TERM) is a developing field that focuses on the advancement of alternative therapies for tissue and organ restoration. Collagen scaffold biomaterials play a vital role as a scaffold to promote cell growth and differentiation to promote the repair and regenerate the tissue lesion. The goal of this chapter will be to evaluate the role of supercritical carbon dioxide extraction technology in the production of collagen scaffold biomaterials from various tissues and organs and relate it to the traditional decellularization techniques in the production of collagen biomaterials for TERM. Therefore, we will study the collagen scaffold biomaterials produced using supercritical carbon dioxide extraction technology and their characteristics, such as chemical-physical properties, toxicity, biocompatibility, in vitro and in vivo bioactivity that could affect the interaction with cells and living system, relative to traditional decellularization technique-mediated collagen scaffolds. Furthermore, the chapter will focus on supercritical carbon dioxide extraction technology for the production of collagen scaffolds biomaterial appropriate for TERM

Development of Tissue Engineered Scaffolds for Cardiovascular Repair and Replacement in Pediatric Patients

Congenital Heart Diseases (CHD) are abnormalities present in the heart and great vessels at birth. It is one of the most frequently diagnosed congenital disorders, affecting approximately 40,000 live birth each year in the United States. The incidence of new CHD patients and the relative distribution of defects have not changed over time and remain a birth rate function. Out of the new patients found to have CHD each year, an estimated 2,500 patients have a defect that requires a substitute, non-native valve, or conduit artery to replace structures that are congenitally absent or hypoplastic. Materials in current use for conduit and valve replacement involve varying degrees of stiffness and flexibility, durability, calcification, susceptibility to infection, thrombosis, and a lack of growth potential for the replacement. Biomaterials developed using tissue engineering principles could overcome the limitations encountered with current strategies. This research aims to develop potentially superior valves and conduits using acellular xenograft tissues that are physically cross- linked to protect the Extracellular Matrix (ECM) from rapid degradation. The hypothesis is that such a replacement graft would allow cellular ingrowth of host cells and potentially enable regenerative growth and remodeling of the graft. A decellularization protocol was developed, and the most effective crosslinker protecting the extracellular matrix structure was identified. The decellularized scaffolds crosslinked with Penta galloyl glucose (PGG) were analyzed in-vitro for stability and mechanical properties, in subcutaneous rat and in valve replacement in sheep-models to determine the biocompatibility and functionality of the developed scaffolds. Tissue-engineered scaffolds prepared from decellularized PGG treated tissues were found to have mechanical properties comparable to that of native tissues, while being more resistant to enzymatic degradation. Subcutaneous implantation of scaffolds demonstrated their biocompatibility and superior resistance to calcification compared to currently available glutaraldehyde fixed tissues. The tissue-engineered conduits and valves implanted in large animal models also demonstrated adequate implant functionality, cellular infiltration, implant remodeling, and growth of the implants. PGG treated decellularized xenografts could be an effective replacement option for pediatric patients, reducing the need for reoperations required with current devices

Mechanisms of Biomaterial-Mediated Cardiac and Esophageal Repair

Biologic scaffolds derived from mammalian extracellular matrix (ECM) have been extensively used in pre-clinical and clinical applications to promote constructive tissue remodeling in a number of anatomic locations. The clinical success of these technologies depends on a number of factors including the species and tissues from which they are derived, the efficacy of the decellularization process, and post-processing modifications such as crosslinking and solubilization, among others. The ECM is produced by the resident cells of every tissue and hence, it is thought to constitute the ideal substrate for each unique cell population. It is therefore logical to assume that a substrate composed of site-specific ECM would be favorable for clinical use in homologous anatomic locations. However, the advantages of using site-specific (homologous) ECM scaffolds in clinical applications is still a matter of debate. Part of the difficulty in addressing this issue arises from the fact that most studies have investigated the application of ECM-derived scaffolds in either homologous or non-homologous locations independently, but they have rarely been directly compared in properly designed studies. The present dissertation shows the development of ECM-based biomaterials derived from cardiac and esophageal tissues. The decellularized scaffolds are compliant with decellularization standards and are then used to evaluate the tissue specific effects of homologous ECM in vitro and in a preclinical models of cardiac and esophageal repair