Dissertations / Theses on the topic 'Tissue engineering. Regenerative Medicine'

Regenerative medicine is an emerging field that aims to treat injury and disease by harnessing and augmenting the body’s innate capacity for tissue regeneration. Many of the strategies developed in this field have relied extensively on the principles of tissue engineering, a set of methods that bring together cells, cellular signals and material scaffolds to repair or replace biological tissue. While the number of novel tissue engineering strategies continues to rapidly expand, the innovations underlying these solutions often fail to consider the key technical, manufacturing, and regulatory barriers that prohibit these technologies from suitable use in humans. As a result, the field of tissue engineering has one of the lowest rates of clinical translation amongst medical research. To address this, this thesis examines each of the prominent components of the tissue engineering practice and develops tools and strategies that enable the development of solutions with high translational potential. The collective findings of these works propose tools and solutions applicable within the major facets of tissue engineering that may help to lay the groundwork for future therapies with high clinical probability in a number of regenerative medicine applications.

Due to the limited regenerative capacity of the central nervous system (CNS) upon injury, regenerative medicine and tissue engineering strategies show great promise for treatment. These aim to restore tissue functions by combining principles of cell biology and engineering, using biomaterial scaffolds which can help in recapitulating the 3D environment of the brain and improving cell survival after grafting. Stroke and TBI are severe forms of disruptions of brain architecture, and two of the leading causes of mortality and morbidity worldwide, as no effective treatments are available. Several studies report how neural stem cells (NSCs) are able to improve functional recovery upon transplantation. However, the efficacy of these treatments is limited because of the mortality these cells are subject to after transplantation. In this context, the transplantation of mesenchymal cells (MSCs) has shown beneficial effects by secreting molecules and factors that help in the healing process. In this study, we tested alginate-based hydrogels as candidates to support human NSCs and MSCs transplantation into the brain, in the view of exploiting the beneficial effects of both and analyzing whether their combined use could have a synergistic effect. In the first part, we studied the suitability of alginate-based scaffolds for the three-dimensional encapsulation and culture of hNSCs and hMSCs. We analyzed their ability to support cell survival, and we evaluated whether changes in their concentration or modifications with ECM molecules could influence cell viability. We showed that the best survival conditions are found when using an RGDs-functionalized alginate scaffold at a low concentration (0.5% w/v). We then worked on the identification of the best conditions for MSCs culture and the definition of coculture conditions. Since serum is necessary for MSCs, but it is reported to induce glial differentiation of NSCs, we explored two different experimental setups. On one hand, we investigated the feasibility to exploit biomaterials to create "compartmentalized" cocultures that would at least partially retain serum. In parallel, we positively observed that MSCs can survive, proliferate and maintain their stemness even in absence of serum, supporting the hypothesis that the use of “compartmentalized” coculture systems would likely be exploitable for MSCs culture. Finally, we tested the reported beneficial effects of MSCs in our 3D culture system, in which NSCs do not show a great viability. Encapsulated NSCs were cultured on an MSCs monolayer, and we analyzed cell survival, proliferation, differentiation and stemness retention. Gene expression analyses highlighted that NSCs maintain stemness characteristics, but we were not able to observe any improvement in NSCs survival in coculture, with respect to standard culture. In the last part of the project we decided to test our system for tissue engineering approaches, exploiting axotomized brain organotypic slices (OSCs). We evaluated the presence of cells 7 days after transplantation, their integration in the OSCs and glial response. Preliminary results suggest that the biomaterial does not cause activation of glial cells, although stem cells do not seem to migrate out of scaffold and integrate into the brain slice.

Due to the limited regenerative capacity of the central nervous system (CNS) upon injury, regenerative medicine and tissue engineering strategies show great promise for treatment. These aim to restore tissue functions by combining principles of cell biology and engineering, using biomaterial scaffolds which can help in recapitulating the 3D environment of the brain and improving cell survival after grafting. Stroke and TBI are severe forms of disruptions of brain architecture, and two of the leading causes of mortality and morbidity worldwide, as no effective treatments are available. Several studies report how neural stem cells (NSCs) are able to improve functional recovery upon transplantation. However, the efficacy of these treatments is limited because of the mortality these cells are subject to after transplantation. In this context, the transplantation of mesenchymal cells (MSCs) has shown beneficial effects by secreting molecules and factors that help in the healing process. In this study, we tested alginate-based hydrogels as candidates to support human NSCs and MSCs transplantation into the brain, in the view of exploiting the beneficial effects of both and analyzing whether their combined use could have a synergistic effect. In the first part, we studied the suitability of alginate-based scaffolds for the three-dimensional encapsulation and culture of hNSCs and hMSCs. We analyzed their ability to support cell survival, and we evaluated whether changes in their concentration or modifications with ECM molecules could influence cell viability. We showed that the best survival conditions are found when using an RGDs-functionalized alginate scaffold at a low concentration (0.5% w/v). We then worked on the identification of the best conditions for MSCs culture and the definition of coculture conditions. Since serum is necessary for MSCs, but it is reported to induce glial differentiation of NSCs, we explored two different experimental setups. On one hand, we investigated the feasibility to exploit biomaterials to create "compartmentalized" cocultures that would at least partially retain serum. In parallel, we positively observed that MSCs can survive, proliferate and maintain their stemness even in absence of serum, supporting the hypothesis that the use of “compartmentalized” coculture systems would likely be exploitable for MSCs culture. Finally, we tested the reported beneficial effects of MSCs in our 3D culture system, in which NSCs do not show a great viability. Encapsulated NSCs were cultured on an MSCs monolayer, and we analyzed cell survival, proliferation, differentiation and stemness retention. Gene expression analyses highlighted that NSCs maintain stemness characteristics, but we were not able to observe any improvement in NSCs survival in coculture, with respect to standard culture. In the last part of the project we decided to test our system for tissue engineering approaches, exploiting axotomized brain organotypic slices (OSCs). We evaluated the presence of cells 7 days after transplantation, their integration in the OSCs and glial response. Preliminary results suggest that the biomaterial does not cause activation of glial cells, although stem cells do not seem to migrate out of scaffold and integrate into the brain slice.

Thesis (Ph. D. in Biomedical Engineering)--Harvard-MIT Program in Health Sciences and Technology, 2013.
Vita. Cataloged from PDF version of thesis.
Includes bibliographical references (pages 85-97).
Anterior cruciate ligament (ACL) injuries affect over 200,000 Americans yearly, and many occur in young athletes. Current treatment options include tendon autografts and cadaveric allografts. However, these approaches often lead to secondary medical problems, such as donor-site morbidity and immune rejection. Furthermore, in younger patients these grafts fail to grow, leading to additional complications and underlining the need for the development of new approaches that improve the healing and repair of ligaments and tendons. This thesis aims to develop a technique to engineer ACL from autologous mesenchymal stem cells (MSC) and primary ACL fibroblasts using the basic principles of Tissue Engineering. The first part of the thesis characterizes MSCs isolated from tibial bone marrow as an alternative to hip-derived marrow aspirates. The proximity of the tibia to the surgical site of ACL reconstructions makes it a viable source of marrow derived-MSCs for ligament repair, with less stress for the patient and increased flexibility in the operating room. Characterization was performed by fluorescenceactivated cell sorting for MSC-surface markers, and assays to differentiate MSCs towards adipogenic, osteogenic and chondrogenic lineages. The second part of the thesis describes the effects of in vitro co-cultures of ACL fibroblast and MSC on the expression of ligament-associated markers. The goal was to optimize the cell-cell ratio in order to maximize the positive effects of co-cultures on ligament regeneration. Co-cultures of ACL fibroblasts and MSCs were studied for 14 and 28 days in vitro, and the effects assessed with quantitative mRNA expression and immunofluorescence of ligament markers Collagen type I, Collagen type III and Tenascin-C. Finally, based on the enhancing effect observed in co-cultures, the thesis explores a method to regenerate ACL using a three-dimensional polyglyconate scaffold seeded with cell-hydrogel suspensions containing ACL fibroblasts and MSCs. Constructs were analyzed biochemically and by immunofluorescence after 4 weeks in culture with and without mechanical stimulation. Together, our results establish an experimental framework from which a new technique for ACL repair can be developed. The ultimate goal is to foster the design of a one-stage surgical procedure for improved primary ACL augmentation repair that can soon be translated into clinical practice.
by José Antonio Canseco.
Ph.D.in Biomedical Engineering

Existing, dermal, regenerative scaffolds facilitate dermal repair and wound healing of severe burn injuries; however, new tissue is often functionally, mechanically and aesthetically abnormal due to irregular deposition of new extracellular matrix. In the present study two novel, elastin-containing scaffolds were developed, characterised and examined both in vitro and in vivo aiming to minimise wound contraction, improve scar appearance and increase skin elasticity post-healing. The first types of scaffolds were electrospun from a triple polymer solution of collagen, elastin and poly(ϵ-caprolactone) (CEP). Two scaffolds were chosen for characterisation: CEP 1 was fabricated using a 1.5 % (w/v) collagen, 12 % (w/v) elastin and 1.5 % (w/v) poly(ϵ-caprolactone) (PCL) solution, a flow rate of 3 mL/h, an air gap of 15 cm and an applied electric potential of 25 kV; and CEP 2 was electrospun using a 2 % (w/v) collagen, 12 % (w/v) elastin and 1 % (w/v) PCL solution at 1 mL/h, 20 cm and 20 kV. In vitro cell studies using human, dermal fibroblasts (HDFs) and immortalised, human keratinocytes (HaCaTs) revealed CEP 1 and CEP 2 supported cell-seeding and cell proliferation with significantly higher proliferation of both cell types on CEP 1. Additionally, subcutaneous implant studies in mice revealed minimal inflammation in response to both scaffolds with CEP 1 vascularised by week 2 post-surgery. However, CEP 1 was rapidly biodegraded after 2 weeks. Collagen deposition was observed in encapsulating tissue and new tissue with consistent collagen expression over 24 weeks. The second type of scaffold investigated was an elastin-modified version of the commercial, dermal substitute Integra Dermal Regeneration Template (IDRT). Elastin-IDRT (EDRT) was developed by inclusion of 10% human tropoelastin and then investigated in comparison with IDRT. Morphological analysis by scanning electron microscope and mechanical characterisation revealed EDRT had significantly enlarged pores, higher porosity and increased deformability. Higher cell seeding efficiency of HaCaTs on EDRT was observed compared to IDRT but cell proliferation rate was found to be similar over 28 days. HDFs displayed increased cell growth rate on EDRT over 28 days compared to IDRT. Enhanced and accelerated HDF infiltration of EDRT was also visualised with complete infiltration by day 14 post-seeding. An in vivo, mouse, subcutaneous implant model showed that EDRT induced minimal inflammation. Gene expression of mouse collagen was consistent over 24 weeks with non-significant increases in elastin expression from weeks 2 and 4. One-step grafting demonstrated similar contraction between EDRT-, IDRT- and autografted wounds with final contraction around 40 % compared to 100 % in open wounds. EDRT displayed significantly accelerated, early-stage angiogenesis with higher vascularisation than IDRT-grafted, autografted or open wounds 2 weeks post grafting. By week 4 EDRT- and IDRT-grafted wounds had similar levels of vascularisation which were higher than autografted and open wounds. EDRT showed improved mechanical performance, supported enhanced cell interactions in vitro and accelerated angiogenesis in vivo. In summary, investigated scaffolds demonstrated properties that could potentially improve burn wound healing. The inclusion of elastin in scaffolds produced by either electrospinning or lyophilisation improved HDF infiltration and supported formation of a confluent layer of HaCaTs which could result in increased pliability of new skin and accelerated wound healing. In EDRT elastin improved scaffold porosity, pore size and accelerated angiogenesis in vivo indicating EDRT can facilitate and improve wound remodelling. Further investigation of both scaffolds is warranted especially due to the vascular inductive effects of EDRT and the synchronous spatial and temporal biodegradation of CEP 2 observed in vivo.

This thesis describes the development and utilisation of a self-reporting scaffold to improve current monitoring methods of the cellular microenvironment. In vitro tissue models hold a lot of promise for regenerative medicine and tissue engineering. However, many models lack the ability to non-invasively monitor in situ cellular responses in a physiologically relevant environment. By development of electrospun self-reporting scaffolds and incorporation of flow culture conditions, this limitation can be overcome. Electrospun matrices have been shown to mimic the structural architecture of the native extracellular matrix, whilst flow conditions have been shown to regulate cellular processes, and enhance mass transport and nutrient exchange throughout polymeric scaffolds. Here we show the development of optically transparent self-reporting electrospun scaffolds that incorporate ratiometric pH-sensitive nanosensors and respond to biological and mechanical cues of the native extracellular matrix through exposure to shear stress. Optically transparent self-reporting scaffolds were fabricated by directly electrospinning pH responsive, ratiometric nanosensors within a gelatin biopolymer matrix. The sensors consist of a porous polyacrylamide matrix which encapsulates pH-sensitive fluorophores that exhibit an additive fluorescent response across the full physiological range between pH 3-8, and a pH-insensitive reference fluorophore. The self-reporting scaffold was able to support cell growth whilst being able to simultaneously monitor local pH changes in real time. A Quasi-Vivo® bioreactor system was also used to generate a flow of cell culture medium and expose cell-seeded scaffolds to a continual shear stress. This novel diagnostic scaffold and the use of flow conditions can help simulate enhance the understanding of in vitro conditions, and generate advanced simulations in vivo to facilitate tissue engineering and regenerative medicine applications.

Anterior cruciate ligament (ACL) ruptures and tears are significant orthopedic problems that result in discomfort and limited mobility. Fully functional tissue engineered ligament replacements are promising alternatives to current graft choices for repair of ACL disruptions. The cell-based approach to construct engineered ligament grafts presented herein involves the culture of mesenchymal stem cells (MSC) on biodegradable, fibrous polymeric scaffolds to promote tissue formation. Multipotent MSCs are advantageous because of their in vitro proliferative capacity and ease of harvest; however; the promotion of MSC differentiation into mature fibroblasts and subsequent extracellular matrix (ECM) development is unknown. The proposed studies utilized three complementary methods to promote differentiation of MSCs: scaffold architecture, mechanical stretch and over-expression of the transcription factor, scleraxis. First, elastomeric scaffolds were fabricated by electrospinning a segmented poly(esterurethane urea) with variations in fiber diameter and fiber alignment. Primary mesenchymal stem cells and the mesenchymal stem cell line, C3H10T1/2, were seeded on these scaffolds and assumed spindle-shaped morphologies and oriented with the direction of fiber alignment. Fiber diameter affected cellular responses, including the expression of ECM genes (e.g. collagen type 1 and decorin) which were elevated on smaller mean fiber diameter scaffolds initially. However, scleraxis gene expression was greatest on larger mean fiber diameter scaffolds at the end of two weeks. Second, cyclic stretch was applied to C3H10T1/2 cells on semi-aligned scaffolds using a novel bioreactor. Cell attachment was verified during and after the application of mechanical stress by confocal microscopy. Cyclic stretch induced cells to assume a highly elongated morphology; however ECM gene expression changes were moderate. Third, forced constitutive expression of scleraxis was accomplished by nucleofection of C3H10T1/2 cells. Transient mRNA expression, accumulation of the gene product in the cell nucleus, and cell death were observed. Future work will seek to refine the experimental methods, including the development and testing of an inducible scleraxis transgene and the application of longer periods of mechanical stimulation. Finally, these complementary approaches may be combined to further extend this work in pursuit of directed differentiation of stem cells and the ensuing generation of a robust tissue graft.
Ph. D.

Bone is a highly organised and specialised connective tissue that provides a rigid, protective and supporting framework to the body. In addition to this, bone is unique in its capacity to self-regenerate without the formation of a fibrotic scar. Despite its natural healing potential, bone is not always able to repair large defects, which can result in permanent bone loss and fracture non-unions. Consequently, interventions such as bone grafting are required to replace damaged or diseased bone, accounting for more than two million grafted bones in the world annually. Currently, autografts are still qualified as the gold standard technique, but this option is not exempt of complications such as infections and donor site morbidity. Advanced therapies (AT), particularly regenerative medicine (RM) and tissue engineering (TE) approaches, provide valuable tools with broad applicability in the orthopaedic field with the aim of achieving bone regeneration. This PhD project was developed within the RM field aiming to optimise the formulation of tissue engineering products (TEPs) composed of mesenchymal stromal cells (MSCs) for bone regeneration. To date, MSC-based therapies have been demonstrated safe and some initial signs of efficacy have already been found in several clinical indications; that is why current major challenges rely on improving the efficacy of such therapies by modifying formulations paying special attention to the tissue source of MSCs as well as to the non-cellular components of the final TEPs. The proposal reported in this PhD project is based on the use of MSCs derived either from bone marrow (BM) or the Wharton’s jelly (WJ) of the umbilical cord (UC) as the osteogenic component of the TEP, decellularised bony particles providing osteoinductive and osteoconductive cues and a hydrogel made of fibrin which confers the ability of adapting to the architecture of each particular defect. The investigation has been performed in vitro and in vivo in an ectopic mice model (addressed in CHAPTER IV) and subsequently in two orthotopic ovine models (addressed in CHAPTER III and CHAPTER V) demonstrating an excellent safety profile and signs of efficacy. The new BM-derived MSCs-based clinical grade formulation developed in this work resulted feasible, effective and efficiently adapted to the architecture of simulated cylindrical bone defects. On the other hand, this work is a milestone in the non-clinical development of WJ-MSCs-derived TEPs prior to use in patients. Nonetheless, further investigations in order to trigger the osteogenic commitment of WJ-derived MSCs for specific bone regeneration indications are required. In addition, the outcomes relating to the injectable bone formulation make it an attractive alternative to be considered in future TE approaches regarding three dimensional (3D) bioprinting, as a potential MSC-based bioink.
El hueso es un tipo de tejido conjuntivo altamente especializado y organizado que proporciona una estructura de soporte rígida y protectora. Además, el hueso es único en su capacidad de autoregeneración sin la formación de una cicatriz fibrótica. A pesar de su potencial regenerador natural, el hueso no es siempre capaz de reparar grandes defectos por sí solo, lo que puede resultar en pérdidas óseas permanentes o en la aparición de pseudoartrosis. Por consiguiente, se requieren intervenciones quirúrgicas para la aplicación de injertos con la finalidad de reemplazar hueso dañado o enfermo. Esto se traduce en la implantación de más de dos millones de injertos óseos anuales en el mundo. Actualmente, los autoinjertos siguen siendo la técnica quirúrgica estándar, pero no están exentos de complicaciones, tales como infecciones o morbilidad asociada a la zona de extracción donante. Las terapias avanzadas (AT), particularmente las aproximaciones dentro de la medicina regenerativa (RM) y la ingeniería de tejidos (TE), ofrecen herramientas valiosas con amplia aplicabilidad en el mundo de la ortopedia con el objetivo de lograr regenerar hueso. Esta tesis doctoral se ha desarrollado dentro del campo de la RM con el objetivo de optimizar la formulación de productos de ingeniería de tejidos (TEPs) compuestos por células mesenquimales estromales (MSCs) con la finalidad de regenerar hueso. Hasta la fecha, se ha acumulado amplia experiencia, tanto preclínica como clínica, demostrando la seguridad e indicios de eficacia de las terapias basadas en el uso de MSCs para diversas indicaciones. Por este motivo, los principales retos en la actualidad se centran en mejorar la eficacia de dichos productos modificando sus formulaciones y prestándole especial atención tanto al tejido de aislamiento de las MSCs como a los componentes no celulares de los TEPs. La propuesta presentada en esta tesis doctoral está basada en MSCs derivadas de médula ósea (BM) o de gelatina de Wharton (WJ) del cordón umbilical (UC) como componente osteogénico, partículas de hueso descelularizadas que aportan las propiedades osteoinductoras y osteoconductoras y un hidrogel de fibrina que confiere la habilidad de adaptarse a la arquitectura de cada defecto en particular. La investigación se ha realizado tanto in vitro como in vivo en un modelo ectópico en ratón (abordado en el CHAPTER IV) y subsecuentemente, en dos modelos ortotópicos en oveja (abordado en el CHAPTER III y en el CHAPTER V) demostrando seguridad y signos de eficacia. La nueva formulación de grado clínico basada en MSCs derivadas de BM resultó ser factible, eficaz y eficiente adaptándose a la arquitectura de los defectos cilíndricos simulados. Por otro lado, este trabajo es un hito en el desarrollo no clínico de los TEPs basados en MSCs derivadas de WJ antes de su aplicación en pacientes. No obstante, se requieren más estudios con el objetivo de desencadenar la rápida diferenciación hacia linaje osteogénico de las MSCs derivadas de WJ en indicaciones específicas de regeneración ósea. Además, los resultados relacionados con la formulación de hueso inyectable la convierten en una alternativa atractiva para ser considerada en futuras aproximaciones de TE relacionadas con la bioimpresión en tres dimensiones (3D), como una potencial biotinta basada en MSC.

Abstract Drug delivery system (DDS) technology is particularly promising to improve the in vivo efficiency of active molecules. Moreover, it is possible to stabilize and prolong the half life at biologically active molecules, thus prolonging the in vitro activity. DDS's can be used either for the delivery of anticancer drug but also for the controlled release of growth factors essential for the tissue engineering. In this work DDS’s have been investigate to develop new targeted anticancer drug or to control the release of growth factor for tissue engineering. In the first case polymer conjugates were chosen while in the second microspheres were used. PEG conjugates Anti-cancer drugs are very active molecules but they present limits that often prevent their success in chemotherapy. Common problems are a low half-life due to rapid kidney clearance, rapid inactivation by metabolic enzymes and low selectivity towards cancer cells and often a low solubility in water, these causing severe side effects. To overcome this problems, polymeric conjugates were prepared by linking an anti-cancer drug to a polymer carrier. Polymeric conjugation improves the drug pharmacokinetic profiles by reducing drug clearance. Furthermore, a tumor targeting can be reached by two mechanisms: the first a passive accumulation into tumour tissue, known as the EPR (Enhanced Permeability and Retention) effect and the second an active targeting when a targeting molecule also is coupled to the conjugate. In this work an heterobifunctional poly-(ethylene glycol) was coupled to both epirubicin (EPI), an anti-cancer drug, and to folic acid (FOL) as targeting residue. The biological activity of the derivate FOL-PEG-EPI was studied in two different culture systems; the classic bi-dimensional (2D) system and the tri-dimensional (3D) system using Puramatrix hydrogel. The last should recreate an environment similar to the in vivo situation. The cytotoxicity activity studies were carried out on the following cell lines; HT-29, expressing a normal level of folic acid membrane receptor (FR), MCF-7, medium FR expression and KB-31cells over-expressing FR. The FOL-PEG-EPI cytotoxicity study, showed higher toxicity in KB-31 cells than MCF-7 and HT-29 in both 2D and 3D cell culture systems. Moreover, the use of the 3D culture system, displayed clearly that FOL-PEG-EPI had selective activity on cells over-expressing the folic acid receptor (KB- 31) compared to HT-29cells where to obtain the IC50 was used a conjugate concentration 3 fold higher than the maximum one permitted in clinic. The uptake of conjugates and epirubicin were studied by flow cytometry, and confocal microscopy. In the first case the cytometry showed the fluorescence signal inside the cells for both FOL-PEG-EPI and epirubicine alone. Also, the confocal analysis have confirmed the internalization, displayed the epirubicin in the nuclei and the conjugate in perinuclear side. Microspheres Tissue engineering is based on various disciplines such as medicine, biology, engineering and chemistry fused to the common aim to obtain or replace organs, or parts of organs in the human body. In general, a construct of tissue engineering is formed by the cellular component and a basic structure with function of support. The cells need to be stimulate by proper growth factor to generate a functional tissue. When the solution of growth factors is injected into the site for regeneration, the biological effect is not always optimal, because the biologically active substances are spread away from the site of action very quickly, or because they cannot get to the targeting site. It is therefore essential to develop a technology that stabilizes the administration and the answer can be a DDS. Therefore, the second part of this study was focused on tissue engineering of bone tissue. The research involved the use a the drug delivery system for the controlled release of TAT-OP1 protein, which stimulates osteogenic differentiation. Osteogenic protein-1 (OP-1 or BMP-7) is a member of Bone Morfogenic Proteins’s family (BMPs). BMPs are a group of multi-functional growth factors belonging to the transforming growth factor β (TGF-β) superfamily. They are implicated in a variety of functions such as the formation of cartilage and bone, and the development of non-osteogenic tissues. BMPs are secreted as a precursor approximately four times longer than the mature form and share a C-terminal distinctive pattern (..C…CXGXC…CC…CXCX..) containing seven cysteines which are the active region of the proteins. In this study we used a recombinant fusion protein called TAT-OP1 which includes a TAT sequence, an Arg rich peptide, derived from a HIV protein, which allows the internalization. The construct TAT-OP1 has 162 amino acids starting with an N-terminal 6His-tag followed by the TAT sequence, a peptidase specific cleavage site (spanning 6 AA) and the C-terminal OP-1 domain (126 AA) containing the cysteines motif. When this type of bioactive molecules is injected directly on the action site, it undergo a rapid inactivation and dilution effect, therefore this is the limit for in vivo use. To avoid this problem, the protein was encapsulated in polylactidecoglycolide (PLGA) microspheres. The bioactive molecules released from microspheres can be easily modulated by setting the formulation parameters and production technique. The spray drying technique was used to obtain the TAT-OP1 microspheres. The microspheres release of the TAT-OP1 over a period of 7 days was 98% and the encapsulation efficiency was 35%. The size measurement by SEM was 0.2-2 μm. The biological activity study on TAT-OP1 microspheres was conducted using pre-osteoblast MC3T3-E1 cell line at two different concentrations, 200nM and 27 nM. After the 7 and 14 days of treatment the cultures showed matrix mineralization and the assay testing for the alkaline phosphatase was positive. Also the presence of characteristic osteogenic markers, such as osteopontin and osteocalcin, was verified by immunofluorescence. These positive results led us to evaluate the biological activity of TAT-OP1 microspheres in a tri-dimensional culture system on cells isolated from umbilical cord blood (UCBMSC). The 3D model was made by using synthetic Puramatrix TM Hydrogel which is able to mimic the natural microenvironment. Following encapsulation of the TAT-OP1 microspheres or the free TAT-OP1 into Puramatrix Hydrogel TM, the cellular response to TAT-OP1 stimulation was evaluated using transmission electron microscopy (TEM) analysis to detect the production of bone-matrix. After 27 days of stimulation with TAT-OP1 loaded microspheres(200 nM), partially aggregated microfibrils were observed around the cells. Calcification deposit and hydroxyapatite crystals were detected only in the cultures treated with TAT-OP1 PLGA microspheres (200nM) controlled release system. Therefore the controlled release of TAT-OP1 from PLGA microspheres was verified to increase the stimulation effectiveness. Future investigations will be directed to further confirm the suitability of this approach to improve the in vitro osteogenic differentiation and the biological activity of TAT-OP1 for an eventual clinical application in the field of bone tissue engineering.
Riassunto I sistemi di drug delivery (DDSs) rappresentano una tecnologia particolarmente promettente per migliorare l'efficacia in vivo e in vitro di molecole biologicamente attive con l’obiettivo di circoscriverne l’effetto su una determinata tipologia di cellule, migliorarne l’efficacia, prolungarne il periodo di emivita e ridurre la tossicità di una terapia. In questo lavoro sono stati studiati due modelli di Drug Delivery: il primo riguarda lo sviluppo di nuovi farmaci antitumorali selettivi mediante un coniugato polimerico, mentre il secondo modello, che trova applicazione nell’ambito dell’ingegneria tissutale, riguarda il rilascio controllato di fattori di crescita mediante microsfere. PEG coniugato I problemi più comuni riguardanti i farmaci anti-tumorali possono essere dovuti ad un tempo di emivita basso a causa di clearance renale rapida, all'inattivazione rapida da parte di enzimi, alla scarsa selettività cellulare e spesso ad una scarsa solubilità in ambiente fisiologico, oltre a gravi effetti collaterali. Per cercare di ovviare, almeno in parte, a questi problemi, è stato preparato un coniugato polimerico direzionato al quale è stato legato un farmaco anti-cancro. Il coniugato migliora il profilo farmacocinetico del farmaco riducendo la clearance. Il “selective tumor targeting” può essere attivo o passivo. Il primo riguarda ligandi di recettori associati al tumore, che raggiungono il bersaglio sfruttando l’affinità ligando-recettore. Il secondo sistema può essere ottenuto sfruttando il cosiddetto effetto EPR (enhanced permeability and retention effect) grazie al quale molecole ad alto peso molecolare raggiungono e si accumulano nell’ambiente peritumorale. In questo lavoro è stato utilizzato un poli-(etilenglicole) eterobifunzionale legato ad epirubicina (EPI), un farmaco anti-cancro, e ad acido folico (FOL), come residuo di targeting. L'attività biologica del derivato FOL-PEG-EPI è stata studiata in due diversi sistemi di coltura, il classico sistema bi-dimensionale ed il sistema tri-dimensionale utilizzando Puramatrix hydrogelTM. Quest’ultimo dovrebbe ricreare un ambiente simile a quello in vivo. Gli studi di attività citotossica sono stati effettuati sulle seguenti linee cellulari: HT-29, MCF- 7 e KB-31 che presentano una diversa espressione del recettore di membrana per l’acido folico (rispettivamente normale espressione, medio-alta, alta). Lo studio di citotossicità su FOL-PEG-EPI ha mostrato maggiore tossicità su cellule KB-31, con sovra-espressione del recettore per l’acido folico, rispetto alle cellule MCF-7 e HT-29, sia in colture 2D che 3D. Inoltre, l’utilizzo del sistema di coltura tri-dimensionale ha dimostrato che FOL-PEG-EPI possiede attività selettiva sulle cellule KB-31, rispetto alle cellule HT-29 dove per ottenere l’IC50 è stata utilizzata una concentrazione di coniugato 3 volte più alta della massima utilizzabile in clinica. L’up-take cellulare dei coniugati ed epirubicina sono stati studiati mediante citofluorimetria e microscopia confocale. Nel primo caso, la citofluorimetria ha mostrato la presenza del segnale di fluorescenza all'interno delle cellule sia per FOL-PEG-EPI che per epirubicina. L'analisi di microscopia confocale ha confermato l’internalizzazione, localizzando in zona nucleare il farmaco libero ed in zona perinucleare il coniugato. Microsfere L’ingegneria dei tessuti è un campo interdisciplinare che applica i principi dell’ingegneria e delle scienze della vita allo sviluppo di sostituti biologici per ristabilire, mantenere o migliorare la funzione di tessuti e organi danneggiati. In questa ricerca si fondono discipline di biologia cellulare, ingegneria, scienza dei materiali e chirurgia allo scopo di costruire, mediante la combinazione di cellule, materiali (“scaffold”) e fattori di crescita, nuovi tessuti funzionali. I fattori di crescita possono essere impiegati per riprodurre le condizioni fisiologiche che consentono alle cellule di crescere, moltiplicarsi e differenziarsi nei diversi tipi di tessuti, ma la loro somministrazione rimane ancora una sfida tecnologica a causa della loro breve emivita nonché della loro difficoltà nel raggiungere il sito di targeting. La seconda parte di questo studio ha riguardato lo sviluppo di un sistema di Drug Delivery applicato all’ingegneria tissutale del tessuto osseo. La ricerca ha coinvolto l'utilizzo di un sistema di veicolazione di farmaci per il rilascio controllato della proteina TAT-OP1, che stimola la differenziazione osteogenica. Osteogenic protein-1 (OP-1 o BMP-7) è un membro della famiglia delle proteine morfogeniche dell’osso (bone morphogenic proteins, BMPs). Le BMP vengono riconosciute come fattori di crescita osteoinduttivi, ovvero promotori della formazione di nuovo tessuto osseo e appartengono alla superfamiglia del TGF-β. Le BMP sono secreti come precursori circa quattro volte più lunghi rispetto alla forma matura e possiedono una porzione C-terminale distintiva (pattern .. C ... CXGXC ... CC ... CXCX ..) contenente sette cisteine che costituiscono la regione attiva di queste proteine. In questo studio è stata utilizzata una proteina ricombinante di fusione chiamata TAT-OP1 che comprende una sequenza TAT, un peptide ricco di arginina derivante dall’HIV e che permette l’internalizzazione. Il costrutto TAT-OP1, di 162 aminoacidi, comprende: una porzione N-terminale 6 His-tag seguito dalla sequenza TAT, un sito di cleavage peptidasi-specifico (spanning 6 AA) e il C-terminale con il dominio OP-1 (126 AA) contenente il motivo di cisteine. Quando questo tipo di molecola bioattiva viene iniettato direttamente nel sito di azione, viene sottoposta ad inattivazione e rapida diluizione; questo ne limita l'uso in vivo. Per ovviare al problema sono state impiegate microsfere di poli-lattidecoglicolide (PLGA) per permettere un rilascio controllato di TAT-OP1 con l'obiettivo di mantenere un livello adeguato della proteina per tempi prolungati, migliorandone l’efficienza. Il rilascio delle molecole bioattive può essere facilmente modulato settando i parametri nella formulazione e nella tecnica di produzione. La tecnica dello spray drying è stata utilizzata per ottenere le microsfere con TAT-OP1. Il rilascio dalle microsfere con TAT-OP1 è stato studiato in un periodo di 7 giorni e l'efficienza di incapsulamento era risultata del 35%. Le dimensioni al microscopio a scansione elettronica (SEM) risultavano comprese tra 0,2-2 µm. Lo studio dell’attività biologica su microsfere con TAT- OP1, è stato condotto utilizzando pre-osteoblasti MC3T3-E1 a due diverse concentrazioni, 200 e 27 nM. Dopo 7 e 14 giorni di trattamento, le cellule mostravano presenza di mineralizzazione della matrice, test per la fosfatasi alcalina positivo e presenza di caratteristici marcatori osteogenici, quali osteopontina e osteocalcina. Questi risultati positivi ci hanno portato a valutare l'attività biologica della TAT- OP1 in microsfere in un sistema tri-dimensionale utilizzando cellule staminali mesenchimali isolate dal sangue del cordone ombelicale (UCBMSC). Il modello 3D è stata ottenuto utilizzando la matrice sintetica Puramatrix hydrogelTM, che è in grado di simulare il microambiente fisiologico. A seguito dell’incapsulazione di TAT-OP1 libera o di microsfere con TAT-OP1 in Puramatrix hydrogelTM, la risposta cellulare alla stimolazione di TAT-OP1 è stata valutata grazie all'analisi di microscopia elettronica a trasmissione (TEM) per rilevare la produzione di matrice ossea. Dopo 27 giorni di stimolazione con TAT-OP1 (200 nM), si osservava la presenza di microfibrille parzialmente aggregate attorno alle cellule. Depositi di calcio e cristalli di idrossiapatite sono stati rilevati solo in culture trattate con microsfere a rilascio controllato di TAT-OP1 (200nM). Pertanto, il rilascio controllato di TAT-OP1 da microsfere di PLGA sembra aumentare l’efficacia di stimolazione. Future indagini saranno dirette a confermare ulteriormente la capacità del presente approccio nel migliorare lo studio di differenziamento osteogenico in vitro e l'attività biologica della TAT-OP1 per un eventuale applicazione clinica nel campo dell’ingegneria tissutale dell’osso.

Currently, the only cure for liver failure is orthotopic liver transplantation. However, there are insufficient donor organs available to treat every patient on the transplant list and many die before they are able to receive a liver transplant. The bioartificial liver (BAL) device is a potential extracorporeal treatment strategy utilising hepatocytes or hepatocyte-like cells (HLCs) within a bioreactor to recapitulate normal liver function and therefore ‘bridge’ a patient with liver failure until they receive a transplant. The work in this thesis utilised tissue engineering methods to develop novel approaches to BAL device design through development and characterisation of a polymer membrane scaffold (“PX”) for hollow fibre bioreactor (HFB) culture and a HLC source generated from the transdifferentiation of pancreatic AR42J-B13 (B13) cells. A flat sheet membrane model was used for the development of asymmetrical, hydrophobic polystyrene (PS) phase inversion membranes. Oxygen plasma significantly increased PS membrane surface wettability through addition of oxygen functional groups to create an environment conducive for cell culture. The treated membrane was henceforth referred to as “PX”. The culture medium HepatoZYME+ was investigated for its ability to induce transdifferentiation of B13 cells to HLCs and maintain the hepatic phenotype. Overall, HepatoZYME+-cultured cells experienced viability loss. A diluted version, “50:50”, showed induction of the hepatic markers carbamoylphosphate synthetase-1 (CPS-1) and HNF4α, as well as a change towards a HLC morphology. When using 50:50 as a maintenance medium, transdifferentiated HLCs retained loss of pancreatic amylase and also induction of hepatic markers, with comparable serum albumin secretion to the established Dex + OSM treatment. However, culture viability in 50:50 was still compromised. Therefore, HepatoZYME+ based media were deemed unsuitable for induction and maintenance compared to Dex-based protocols. PX flat sheet membranes were able to support culture of B13 cells and also the human osteosarcoma cell line, MG63, demonstrating improved cell attachment over non-surface treated PS membranes. PX membranes supported transdifferentiation of B13 cells to HLCs, presenting with loss of pancreatic amylase, induction of the hepatic markers transferrin, GS and CPS-1 and serum albumin secretion. Furthermore, PX showed no change in mass or loss of culture surface area over 15 days in culture conditions. Together, the novel membrane material and the media formulation and feeding regime developed have strong potential to be translated to a HFB setting and guide future BAL device design.

Nowadays, there are approximately 10 million people worldwide with visual impairment due to corneal diseases. Currently, the main therapeutic solution is the transplant of a donor's cornea. The great majority of transplants is due to some failure in the inner layer of the cornea, which is called the corneal endothelium and this is mainly related with the inability of this layer to regenerate in vivo. However, transplants present several limitations such as the low number of healthy donors or immunological rejection by the patient. In order to overcome these problems, several researchers have focused in culturing corneal endothelial cells (CEC) to subsequently replace non-functional CEC. However, cell therapy is still very recent and still presents a series of drawbacks. For instance, using animal CEC or cells from other patients has shown to lead into immunological rejection. In order to avoid this, it is possible to use stem cells from the same patient, which have the ability to differentiate into many cell types, including the corneal endothelium. Currently, the stem cells used to regenerate CEC are mainly pluripotent stem cells, either embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC), which are derived from adult cells. Despite their great potential for treating diseases, these types of stem cells present major limitations such as the risk of teratoma formation. In addition, they present other disadvantages such as ethical problems associated with the use of ESCs, safety problems related to iPSC since they requires the use of virus for their production hence limiting its clinical application. For this reason, and in order to solve the current problems in the regeneration of corneal endothelium, this thesis project uses dental pulp stem cells (DPSC) for the formation of CEC. DPSC are an accessible source derived from the same patient, avoiding possible future problems of rejection. In addition, the use of DPSC avoids the ethical and security problems associated with ESC and iPSC. Furthermore, DPSC and CEC have the same embryological origin, as they both arise from neural crest stem cells. In fact, DPSC express neural crest stem cells markers, which facilitates their differentiation into neural crest stem cells (NCSC), which is an intermediate step for the formation of CEC. Therefore, this thesis project uses a two-step protocol, where DPSC are differentiated into NCSC and, subsequently, NCSC are derived into CEC. Because the use of cell therapies alone may present limited cell viability once it is injected, the field of tissue engineering is a new discipline that has appeared to overcome this limitation. Tissue engineering combines the use of cells, biomaterials and biological molecules. It has been demonstrated that the use of different topographies in cell culture modulates cell behavior, and may have an effect on their functionality, cell distribution or cell size. Therefore, this thesis project applies tissue engineering as another strategy for the generation of functional CEC with its characteristic phenotype and morphology. For doing this, we have mimicked the natural CEC environment by cultivating the cells on substrates with different curvatures, composition or topographies that are able to mimic those of the human eye. In conclusion, this thesis project proposes the use of bioengineering, by differentiating CEC from stem cells derived from the patient and the use of biomaterials with different topographies and curvatures, for the regeneration of corneal endothelium.

Bone tissue engineering is a promising approach to address the limitations of currently used bone tissue substitutes. However, an optimal cell source for the production of osteoblastic matrix proteins and mineral deposition has yet to be defined. In response to this deficiency, ex vivo gene therapy of easily accessible non-osteogenic cells, such as skeletal myoblasts, has become a prevalent strategy for inducing an osteoblastic phenotype. The majority of these approaches focus on constitutive overexpression of soluble osteogenic growth factors such as bone morphogenetic proteins (BMPs). In order to avoid aberrant effects of unregulated growth factor secretion, this work focuses on delivery of the osteoblastic transcription factor Runx2 as an autocrine osteogenic signal under the control of an inducible expression system. The overall objective of this research was to engineer an inducible cell source for bone tissue engineering that addresses the limitations of current cell-based approaches to orthopedic regeneration. Our central hypothesis was that inducible Runx2 overexpression in skeletal myoblasts would stimulate differentiation into a regulated osteoblastic phenotype. We have demonstrated that Runx2 overexpression stimulates transdifferentiation of primary skeletal myoblasts into a mineralizing osteoblastic phenotype. Furthermore, we have established Runx2-engineered skeletal myoblasts as a potent cell source for bone tissue engineering applications in vitro and in vivo, similar to BMP-2-overexpressing controls. Finally, we exogenously regulated osteoblastic differentiation by myoblasts engineered to express a tetracycline-inducible Runx2 transgene. This conversion into an osteoblastic phenotype was inducible, repressible, recoverable after suppression, and dose-dependent with tetracycline concentration. This work is significant because it addresses cell sourcing limitations of bone tissue engineering, develops controlled and effective gene therapy methods for orthopedic regeneration, and establishes a novel strategy for regulating the magnitude and kinetics of osteoblastic differentiation.

This research centers on the fabrication, characterization, and engineering of inverse opal scaffolds, a novel class of three-dimensional (3D) porous scaffolds made of biocompatible and biodegradable polymers, for applications in tissue engineering and regenerative medicine. The unique features of an inverse opal scaffold include a highly ordered array of pores, uniform and finely tunable pore sizes, high interconnectivity, and great reproducibility. The first part of this work focuses on the fabrication and functionalization of inverse opal scaffolds based on poly(D,L-lactic-co-glycolic acid) (PLGA), a biodegradable material approved by the U.S. Food and Drug Administration (FDA). The advantages of the PLGA inverse opal scaffolds are also demonstrated by comparing with their counterparts with spherical but non-uniform pores and poor interconnectivity. The second part of this work shows two examples where the PLGA inverse opal scaffolds were successfully used as a well-defined system to investigate the effect of pore size of a 3D porous scaffold on the behavior of cell and tissue growth. Specifically, I have demonstrated that i) the differentiation of progenitor cells in vitro was dependent on the pore size of PLGA-based scaffolds and the behavior of the cells was determined by the size of individual pores where the cells resided in, and ii) the neovascularization process in vivo could be directly manipulated by controlling a combination of pore and window sizes when they were applied to a mouse model. The last part of this work deals with the novel application of photoacoustic microscopy (PAM), a volumetric imaging modality recently developed, to tissue engineering and regenerative medicine, in the context of non-invasive imaging and quantification of cells and tissues grown in PLGA inverse opal scaffolds, both in vitro and in vivo. Furthermore, the capability of PAM to monitor and quantitatively analyze the degradation of the scaffolds themselves was also demonstrated.

This thesis outlines the development of a mechanically functional tissue-engineered cartilage construct that may assist in the repair of damaged joint surfaces. By integrating 3D-printed scaffolds within engineered cartilage, the thesis demonstrates that it is possible to closely recapitulate the complex mechanical behaviour of native tissue. Furthermore, it is shown that the functionality of engineered cartilage can be further improved by optimisation of scaffold properties and mechanical loading in specialised bioreactor systems. By enabling the manufacture of biologically and mechanically relevant cartilage tissues, this work represents an important step towards the translation of functional tissue engineering principles into clinical practice.

Repair of large bone defects remains a clinical challenge for orthopedic surgeons. Current treatment strategies such as autograft and allograft are limited by the amount of available tissue in the case of the former, and failure of revascularization effecting engraftment in the case of the latter. Tissue engineering offers an alternative approach to this challenging clinical problem. The general principle of tissue engineering for bone regeneration prescribes delivery of osteoinductive factors to induce an endogenous response within the host to repair a defect that will not normally heal. One such tissue engineering approach is cell based therapy and this is attractive in the cases of patients with a lack of endogenous osteoprogenitors cells due to volumetric loss of tissue/ageing. Stem cell therapy has emerged as a possible alternative to current treatment modalities, however many challenges to clinical translation remain. Central to these challenges for bone tissue engineering are lingering questions of which cells to use and how to effectively deliver those cells. The goal of this thesis was to elucidate more effective ways to enhance bone repair utilizing adult stem cells. First, we investigated adipose derived stem cells (ADSCs) as a viable cell source for bone tissue engineering. Upon isolation, adipose derived stem cells are a heterogeneous population of multipotent cells predisposed to adipogenic differentiation. We developed an enrichment protocol that demonstrated the osteogenic potential of ADSCs can be enhanced in a dose dependent manner with resveratrol, which had been demonstrated to up-regulate Runx-2 expression. This enrichment strategy produced an effective method to enhance the osteogenic potential of ADSCs while avoiding cell sorting and gene therapy techniques, thus bypassing the use of xenogenic factors to obtain an enriched source of osteoprogenitor cells. This protocol was also used to investigate differences between human and rat ADSCs and demonstrated that rat ADSCs have a higher osteogenic potential than human ADSCs in vitro. The second major thrust of this thesis was to develop an injectable hydrogel system to facilitate bone formation in vivo. Both a synthetic and a naturally based polymer system was investigated, the results of which demonstrated that the naturally based alginate hydrogel was a more effective vehicle for both cell viability in vitro and bone formation in vivo. Our results also demonstrated that despite the ability to increase the osteogenic potential of ADSCs in vitro with resveratrol treatment, this was insufficient to induce bone formation in vivo. However, the inclusion of bone marrow mesenchymal stem cells (BMMSCs) in BMP-2 functionalized alginate hydrogels resulted in significantly greater mineralization than acellular hydrogels. Finally, the effect of timing of delivery of therapeutics to a non-healing segmental bone defect in the femur was investigated. We hypothesized that delivery of biologics after the initial inflammation response caused by injury to the host tissue would result in greater regeneration of tissue in terms of newly formed bone. Contrary to our initial hypothesis, these experiments demonstrated that delayed implantation of therapeutics has a detrimental effect on the overall healing response. It was, however, demonstrated that the inclusion of BMMSCs results in greater bone volume regenerated in the defect site over acellular hydrogels. In conclusion, this work has rigorously investigated the use of adipose derived stem cells for bone tissue engineering, and further produced an injectable hydrogel system for stem cell based bone tissue engineering. This work also demonstrated that the inclusion of adult stem cells, specifically BMMSCs, can enhance the regeneration response in a non-healing bone defect model relative to acellular hydrogel.

Vat photopolymerization (VP) is an additive manufacturing (AM) technology that permits the fabrication of parts with complex geometries and feature sizes as small as a few microns. These attributes make VP an attractive option for the fabrication of scaffolds for tissue engineering. However, there are few printable materials with low cytotoxicity that encourage cellular adhesion. In addition, these resins are not readily available and must be synthesized. A novel resin based on 2-acrylamido-2-methyl-1-propanesulfonic acid (NaAMPS) and poly(ethylene glycol) diacrylate (PEGDA) was formulated and printed using VP. The mechanical properties, water content, and high fidelity of the scaffold indicated promise for use in tissue engineering applications. Murine fibroblasts were observed to successfully adhere and proliferate on the scaffolds. The growth, migration, and differentiation of a cell is known to dependent heavily on its microenvironment. In engineered constructs, much of this microenvironment is provided by the tissue scaffold. The physical environment results from the scaffold's geometrical features, including pore shape and size, porosity, and overall dimensions. Each of these parameters are known to affect cell viability and proliferation, but due to the difficulty of isolating each parameter when using scaffold fabrication techniques such as porogen leaching and gas foaming, conflicting results have been reported. Scaffolds with pore sizes ranging from 200 to 600 μm were fabricated and seeded with murine fibroblasts. Other geometric parameters (e.g., pore shape) remained consistent between scaffold designs. Inhomogeneous cell distributions and fewer total cells were observed in scaffolds with smaller pore sizes (200-400 μm). Scaffolds with larger pores had higher cell densities that were homogeneously distributed. These data suggest that tissue scaffolds intended to promote fibroblast proliferation should be designed to have pore at least 500 μm in diameter. Techniques developed for selective placement of dissimilar materials within a single VP scaffold enabled spatial control over cellular adhesion and proliferation. The multi-material scaffolds were fabricated using an unmodified and commercially available VP system. The material preferences of murine fibroblasts which resulted in their inhomogeneous distribution within multi-material scaffolds were confirmed with multiple resins and geometries. These results suggest that multi-material tissue scaffolds fabricated with VP could enable multiscale organization of cells and material into engineered constructs that would mimic the function of native tissue.
Doctor of Philosophy
Vat photopolymerization (VP) is a 3D printing (or additive manufacturing) technology that is capable of fabricating parts with complex geometries with very high resolution. These features make VP an attractive option for the fabrication of scaffolds that have applications in tissue engineering. However, there are few printable materials that are biocompatible and allow cells attachment. In addition, those that have been reported cannot be obtained commercially and their synthesis requires substantial resources and expertise. A novel resin composition formulated from commercially available components was developed, characterized, and printed. Scaffolds were printed with high fidelity. The scaffolds had mechanical properties and water contents that suggested they might be suitable for use in tissue engineering. Fibroblast cells were seeded on the scaffolds and successfully adhered and proliferated on the scaffolds. The growth, migration, and differentiation of cells is influenced by the environmental stimuli they experience. In engineered constructs, the scaffold provides many of stimuli. The geometrical features of scaffolds, including how porous they are, the size and shape of their pores, and their overall size are known to affect cell growth. However, scaffolds that have a variety of pore sizes but identical pore shapes, porosities, and other geometric parameters cannot be fabricated with techniques such as porogen leaching and gas foaming. This has resulted in conflicting reports of optimal pore sizes. In this work, several scaffolds with identical pore shapes and porosities but pore sizes ranging from 200 μm to 600 μm were designed and printed using VP. After seeding with cells, scaffolds with large pores (500-600 μm) had a large number of evenly distributed cells while smaller pores resulted in fewer cells that were unevenly distributed. These results suggest that larger pore sizes are most beneficial for culturing fibroblasts. Multi-material tissue scaffolds were fabricated with VP by selectively photocuring two materials into a single part. The scaffolds, which were printed on an unmodified and commercially available VP system, were seeded with cells. The cells were observed to have attached and grown in much larger numbers in certain regions of the scaffolds which corresponded to regions built from a particular resin. By selectively patterning more than one material in the scaffold, cells could be directed towards certain regions and away from others. The ability to control the location of cells suggests that these printing techniques could be used to organize cells and materials in complex ways reminiscent of native tissue. The organization of these cells might then allow the engineered construct to mimic the function of a native tissue.

Severe traumatic injuries and surgical procedures like tumor resection often create peripheral nerve gaps, accounting for over 250,000 injuries in the US annually. The clinical "gold standard" for bridging peripheral nerve gaps is autografts, with which 40-50% of patients regain useful function. However, issues including their limited availability and collateral damage at the donor site limit the effectiveness and use of autografts. Therefore, it is critical to develop alternative bioengineered approaches that match or exceed autograft performance. With the use of guidance channels, the endogenous regeneration process spontaneously occurs when successful bridging of short gaps (< 10mm) occurs, but fails to occur in the bridging of longer gaps (≥15mm). Several bioengineered strategies are currently being explored to bridge these critical size nerve gaps. Other labs and ours have shown how filler materials that provide topographical cues within the nerve guides are able to enhance nerve growth and bridge critical length gaps in rats. However, the mechanism by which intra-luminal fillers enhance nerve regeneration has not been explored. The main goal of this dissertation was to explore the interplay between intra-luminal scaffolds and orchestrated events of provisional fibrin matrix formation, glial cell infiltration, ECM deposition and remodeling, and axonal infiltration - a sequence we term the 'regenerative' sequence. We hypothesized that the mechanism by which thin films with topographical cues enhance regeneration is by serving as physical 'organizing templates' for Schwann cell infiltration, Schwann cell orientation, extra-cellular matrix deposition/organization and axon infiltration. We demonstrate that aligned topographical cues mediate their effects to the neuronal cells through optimizing fibronectin adsorption in vitro. We also demonstrate that aligned electrospun thin films are able to enhance bridging of a critical length nerve gap in vivo by stabilizing the provisional matrix, creating a pro-inflammatory environment and influencing the maturation of the regenerating cable leading to faster functional recovery compared to smooth films and random fibers. This research will advance our understanding of the mechanisms of peripheral nerve regeneration, and help develops technologies that are likely to improve clinical outcomes after peripheral nerve injury.

Although millions of individuals worldwide are affected by blinding retinal degenerative diseases, most have very few options for treatment and no hope for vision restoration. Induced pluripotent stem cell (iPSC) replacement therapies represent a promising treatment option, but their effectiveness is limited by an overall lack of physical support for injected cells. Stem cell scaffolds can be used to provide this support by serving as an attachment platform for cells before, during, and after implantation. Thus, the design of polymer scaffolds with appropriate biochemistry, mechanical properties, and morphology is a critical step toward developing feasible stem cell therapies for blinding eye diseases. In this work, we aim to design a regenerative scaffold for the retina and determine the interplay among these three key design parameters. First, the feasibility of using a synthetic scaffold to grow and differentiate iPSCs to neural progenitor cells is demonstrated. The porous and degradable poly(lactic-co-glycolic acid) scaffolds employed were able to support a greater density of differentiating iPSCS than traditional tissue culture plastic. Additionally, the power of chitosan, a naturally occurring polymer, to overcome the toxic effects of copper nanoparticles is described. For two different cell types, various doses, and several time points, chitosan coated copper nanoparticles were significantly less toxic than non-coated particles. The mechanical properties of the human retina and the effects of aging and disease were also estimated using measurements of compressive modulus in animal models. In order to reach a range similar to native tissue, polymer mechanical properties were controlled using cross-linking density and surfactant templating. The influence of morphology was studied by inducing polymer structure changes via surfactant templating. Morphology significantly influenced water uptake and compressive modulus for both cross-linked poly(ethylene glycol) (PEG) and cross-linked chitosan hydrogels. Surfactant templating did not negatively affect the biocompatibility of PEG hydrogels and slightly improved the ability of chitosan hydrogels to support the growth and differentiation of iPSCs. Overall we have demonstrated the ability to tune polymer structure, mechanical properties, and biochemistry. These results add to the growing body of research aimed to understand and control cell/material interactions for biomaterial optimization.

Current clinical approaches to cartilage repair fail to restore the physiological function of the tissue, prompting the development of alternative strategies. This thesis focuses on advancing technologies to generate tissue-engineered cartilage as biological implants for joint resurfacing. It examines the effects of hydrogel scaffolds and mechanical stimulation facilitated by custom bioreactor systems on the development of ex vivo engineered cartilage tissues. The findings of this thesis demonstrate that hydrogels can be engineered to be tough and cell-instructive to provide mechanical support and promote cartilage growth, which can further be enhanced by biaxial mechanical loading simulating the native biomechanical joint environment.

Tendon and ligament injuries are common orthopedic problems that have an enormous impact on the quality of life of affected patients. Despite the frequency at which these injuries occur, current treatments are unable to restore native function to the damaged tissue. Because of this, reinjury is common. It is well known that mechanical stimulation is beneficial for promoting tendon and ligament development and tissue homeostasis; however, the specific mechanisms remain unclear. The transcription factor scleraxis (Scx) is an interesting candidate for mediating the tendon and ligament mechanoresponse, as it has been shown that Scx expression is induced by cyclic mechanical strain in tenocytes and is required for mechanically-induced stem cell tenogenesis. Moreover, Scx expression is increased in adult tendons following exercise. The studies described in this dissertation therefore focus on the combined role of Scx and mechanical stimulation in two contexts: 1) influencing ligament cell differentiation and 2) regulating adult tenocyte behavior. In the first study, transient Scx overexpression combined with mechanical strain in a 3D collagen hydrogel model was investigated as a means of deriving mature ligament cells from stem cells for use in ligament tissue engineering. Scx overexpression in C3H10T1/2 cells cultured in collagen hydrogels under static strain resulted in increased construct contraction and cell elongation, but no concurrent increase in the expression of ligament-related genes or production of glycosaminoglycans (GAG). When combined with low levels of cyclic strain, Scx overexpression resulted in increased mechanical properties of the tissue constructs, increased GAG production, and increased expression of ligament-related genes compared to cyclic strain alone. Together, these results demonstrate that Scx overexpression combined with cyclic strain can induce ligament cell differentiation and suggest that Scx does so by improving the mechanosensitivity of cells to cyclic strain. In the second study, the role of Scx in adult tenocyte mechanotransduction was explored using RNA-sequencing (RNA-seq) and small interfering RNA (siRNA) technologies. Equine tenocytes were exposed to siRNA targeting Scx or a control siRNA and maintained under cyclic mechanical strain prior to being submitted for RNA-seq. Comparison of the resulting transcriptomes revealed that Scx knockdown decreased the expression of several genes encoding important focal adhesion adaptor proteins. Correspondingly, Scx-depleted tenocytes showed abnormally long focal adhesions, decreased cytoskeletal stiffness, and an impaired ability to migrate on soft surfaces. This suggests that Scx regulates the tenocyte mechanoresponse by promoting the expression of focal adhesion-related genes. Combined, the results of these studies support a role for Scx in tendon and ligament cell mechanotransduction and identify the regulation of genes related to maintaining the cell-extracellular matrix connection and cytoskeletal dynamics as a potential mechanism. These findings enhance our understanding of how mechanical stimulation influences cell behavior and provide new research directions and methodologies for future studies of tendon and ligament mechanobiology.
Ph. D.

The interface between tendon/ligament and bone tissue is a complex transition of biochemical, cellular, and mechanical properties. Investigating computational and tissue engineering models that imitate aspects of this interface may supply critical design parameters for designing future tissue replacements to promote increased biochemical and mechanical integration between tendon/ligament and bone. Strategies for modeling this tissue have typically focused on the development of heterogeneous structures to create gradients or multiphasic materials that mimic aspects of the transition. However, further work is required to elucidate the role of specific mechanical and material stimuli in recapitulating features of the tendon/ligament-bone insertion. In particular, in constructs that exhibit variation in both mechanical and biochemical properties, the interplay of mechanical, material, and chemical signals can complicate understanding of the particular factors at work in interface formation. Thus, the overall goal of this dissertation was to provide insight into the role of mechanical strain and scaffold degradability on cell behavior within heterogeneous biomaterials. Specifically, a method for determining cell vertical position within a degradable gel through a laminated interface was developed. A computational model was created to examine possible variation in local mechanical strain due to heterogeneity in mechanical properties and different interface geometries. Finally, the influence of biomaterial degradability on changes in encapsulated human mesenchymal stem cell morphology under response to cyclic mechanical strain was explored. Together, these studies provide insight into mechanical and material design considerations when devising tissue engineering strategies to regenerate the tendon/ligament-bone interface.

In recent years, the degradation and subsequent loss of tissues is an issue that has affected people worldwide. Although there are treatments addressing the degradation of tissues, such treatments involve complicated and expensive procedures, where full tissue regeneration is not achieved. For these reasons, in recent years, tissue engineering has developed cutting-edge biomaterials capable of inducing effective tissue regeneration both under cellular or acellular conditions. Peptide hydrogels are versatile biomaterials composed of the basic components of life amino acids, which act as building blocks to form hierarchical structures, which subsequently go on to form well-defined scaffolds. Biomaterials have been widely used for the culture of mammalian cells, tissue engineering, regenerative medicine, drug delivery, etc. This is thanks to their capability of providing a three-dimensional architecture to cells, which mimics the natural architecture of the extracellular matrix (ECM). Peptide- based hydrogels can be easily functionalised with active biological cues, which can direct the cellular response. It has been shown that the ionic-complementary FEFEFKFK hydrogel, succeeded to support the culture of mammalian cells such as bovine chondrocytes. In this work, we used the same FEFEFKFK hydrogel to investigate the capability of this hydrogel to support the three-dimensional culture of both human osteoblasts (hOBs), and human mesenchymal stem cells (hMSCs) for bone regeneration applications. To achieve this goal, hOBs were cultured within both FEFEFKFK (non-functionalised) and RGD-FEFEFKFK (functionalised) gels. Then the suitability of the FEFEFKFK gels to induce cellular proliferation, synthesis of bone ECM and mineralisation was explored. In addition, taking advantage of the inherent plasticity of hMSCs, we also investigated the capability of the FEFEFKFK gel to foster the osteogenic differentiation of hMSCs, and subsequently to induce bone mineralisation in 3-D under osteogenic stimulation. Based on the results obtained in this work, the FEFEFKFK gel arises as a promising biomaterial for both bone and dental tissue regeneration applications.

The primary aim of this multidisciplinary project was to develop a new generation of breast implants. Disrupting the currently prevailing paradigm of silicone implants which permanently introduce a foreign body into mastectomy patients, highly porous implants developed as part of this PhD project are biodegradable by the body and augment the growth of natural tissue. Our technology platform leverages computer-assisted-design which allows us to manufacture fully patient-specific implants based on a personalised medicine approach. Multiple animal studies conducted in this project have shown that the polymeric implant slowly degrades within the body harmlessly while the body's own tissue forms concurrently.

Bone defects and fracture non-unions remain a substantial challenge for clinicians due to a high occurrence of delayed union or non-union requiring surgical intervention. The current grafting procedures used to treat these injuries have many limitations and further long-term complications associated with them. This has resulted in research efforts to identify graft substitution therapies that are able to repair and replace tissue function. Many of these tissue engineered products include the use of growth factors to induce cell differentiation, migration, proliferation, and/or matrix production. However, current growth factor delivery methods are limited by poor retention of growth factors upon implantation resulting in low bioactivity. These limiting factors lead to the use of high doses and frequent injections, putting the patients at risk for adverse effects. The goal of this work was to develop and evaluate the efficacy of BMP-2 delivery systems to improve bone regeneration. We examined two approaches for delivery of BMP-2 in this work. First, we evaluated the use of a self-assembling lipid microtube system for the sustained delivery of BMP-2. We determined that sustained delivery of BMP-2 from the lipid microtube system was able to enhance osteogenic differentiation compared to empty microtubes, however did not demonstrate a significant advantage compared to a bolus BMP-2 dose in vitro. Second, we developed and assessed the functionality of an affinity-based system to sequester BMP-2 at the implant site and retain bioactivity by incorporating heparin within a collagen matrix. Incorporation of heparin in the collagen matrix improved BMP-2 retention and bioactivity, thus enhancing cell-mediated mineralized matrix deposition in vitro. Lastly, the affinity-based BMP-2 delivery system was evaluated in a challenging in vivo bone repair model. Delivery of pre-bound BMP-2 and heparin in a collagen matrix resulted in new bone formation with mechanical properties not significantly different to those of intact bone. Whereas delivery of BMP-2 in collagen or collagen/heparin matrices had similar volumes of regenerated mineralized tissue but resulted in mechanical properties significantly less than intact bone properties. The work presented in this thesis aimed to address parameters currently preventing optimal performance of protein therapies including stability, duration of exposure, and localization at the treatment site. We were able to demonstrate that sustained delivery of BMP-2 from lipid microtubes was able to induce osteogenic differentiation, although this sustained delivery approach was not significantly advantageous over a bolus dose. Additionally, we demonstrated that the affinity-based system was able to improve BMP-2 retention within the scaffold and in vitro activity. However, in vivo implantation of this system demonstrated that only delivery of pre-complexed BMP-2 and heparin resulted in regeneration of bone with mechanical properties not significantly different from intact bone. These results indicate that delivery of BMP-2 and heparin may be an advantageous strategy for clinically challenging bone defects.

Severe extremity trauma often involves significant damage to multiple tissue types, including bones, skeletal muscles, peripheral nerves, and blood vessels. Such injuries present unique challenges for reconstruction, and improving structural and functional outcomes of intervention remains a pressing, unmet clinical need. While tissue engineering/regenerative medicine (TE/RM) therapeutics offer promising potential to overcome the status quo limitations of surgical reconstruction, very few products have transitioned to clinical practice. Improving treatment options will likely require advancing our understanding of the biological interactions occurring in the repair of damaged tissues. Bone tissue is known to be innervated and highly vascularized, and both tissue types are involved in normal bone physiology. However, the degree to which these tissue relationships influence the repair of large, multi-tissue defects remains unknown. Accordingly, the goal of this thesis was to investigate tissue regeneration in two novel composite injury models. First, we characterized interactions in a composite bone and nerve injury model where a segmental bone defect was combined with a peripheral nerve gap. Our results indicated that although tissue regeneration was not impaired, the composite injury group experienced a marked functional deficit in the operated limb compared to single-tissue injury. Second, we developed a model of composite bone and vascular extremity trauma by combining a critically-sized segmental bone defect with surgically-induced hind limb ischemia to evaluate the effects on BMP-2-mediated bone repair. Interestingly, our results demonstrated a stimulatory effect of the recovery response to ischemia on bone regeneration. Finally, we investigated early vascular growth and gene expression as potential mechanisms coupling the response to ischemia with bone defect repair. Although the response to ischemia promoted robust vascular growth in the thigh, it did not directly augment vascularization at the site of bone regeneration. In addition, the stimulatory effects of ischemia on bone regeneration could not be explained by gene expression alone based on the genes and time points investigated. Taken together, this thesis presents pioneering work on a new thrust of TE/RM research – tissue regeneration in models of composite injury. This work has provided new insights on the complexity of composite tissue repair, specifically in regard to the relationship between vascular tissue growth and bone healing. Going forward, successful leverage of models of composite tissue injuries will provide valuable test beds for screening new technologies, advance the understanding of tissue repair biology, and ultimately, may produce new therapeutic interventions for limb salvage and reconstruction that improve outcomes for extremity trauma patients.

Tendon function is essential for quality of life, yet the pathogenesis and healing of tendinopathy remains poorly understood compared to other musculoskeletal disorders. The aim of regenerative medicine is to replace traditional tissue and organ transplantation by harnessing the developmental potential of stem cells to restore structure and function to damaged tissues. The recently discovered interdependency of cell phenotype and biophysical environment has created a paradigm shift in cell biology. This dissertation introduces a dynamic in vitro model for tendon function, dysfunction and development, engineered to characterize the mechanobiological relationships dictating stem cell fate decisions so that they may be therapeutically exploited for tendon healing. Cells respond to mechanical deformation via a complex set of behaviors involving force-sensitive membrane receptor activity, changes in cytoskeletal contractility and transcriptional regulation. Effective ex vivo model systems are needed to emulate the native environment of a tissue and to translate cell-matrix forces with high fidelity. A naturally-derived decellularized tendon scaffold (DTS) was invented to serve as a biomimetic tissue culture platform, preserving the structure and function of native extracellular matrix. DTS in concert with a newly designed dynamic mechanical strain system comprises a tendon bioreactor that is able to emulate the three-dimensional topography, extracellular matrix proteins, and mechanical strain that cells would experience in vivo. Mesenchymal stem cells seeded on decellularized tendon scaffolds subject to cyclic mechanical deformation developed strain-dependent alterations in phenotype and measurably improved tissue mechanical properties. The relative tenogenic efficacies of adult stem cells derived from bone marrow, adipose and tendon were then compared in this system, revealing characteristics suggesting tendon-derived mesenchymal stem cells are predisposed to differentiate toward tendon better than other cell sources in this model. The results of the described experiments have demonstrated that adult mesenchymal stem cells are responsive to mechanical stimulation and, while exhibiting heterogeneity based on donor tissue, are broadly capable of tenocytic differentiation and tissue neogenesis in response to specific ultrastructural and biomechanical cues. This knowledge of cellular mechanotransduction has direct clinical implications for how we treat, rehabilitate and engineer tendon after injury.
Ph. D.

Biomimetic materials that recapitulate the complex mechanical and biochemical cues in load-bearing tissues are of significant interest in regenerative medicine and tissue engineering applications. Several investigators have endeavored to not only emulate the mechanical properties of the vasculature, but to also mimic the biologic responsiveness of the blood vessel in creating vascular substitutes. Previous studies in our lab generated the elastin-like protein polymer LysB10, which was designed with the capability of physical and chemical crosslinks, and was shown to display a range of elastomeric properties that more closely matched those of the native artery. While extensive validation of the mechanical properties of elastin-mimetic polymers has demonstrated their functionality in a number of tissue engineering applications, limited cell growth on the surfaces of the polymers has motivated further optimization for biological interaction. Recent biologically-inspired surface strategies have focused on functionalizing material surfaces with extracellular matrix molecules and bioactive motifs in order to encourage integrin-mediated cellular responses that trigger precise intracellular signaling processes, while limiting nonspecific biomaterial interactions. Consequently, this dissertation addresses three approaches to modulating cellular behavior on elastin-mimetic analogs with the goal of promoting vascular wall healing and tissue regeneration: genetic engineering of elastin-like protein polymers (ELPs) with cell-binding domains, biofunctionalization of elastin-like protein polymers via chemoselective ligation of bioactive ligands, and incorporation of matrix protein fibronectin for engineering of cell-seeded multilamellar collagen-reinforced elastin-like constructs. The synthesis of recombinant elastin-like protein polymers that integrate biologic functions of the extracellular matrix provides a novel design strategy for generating clinically durable vascular substitutes. Ultimately, the synthesis of model protein networks provides new insights into the relationship between molecular architecture, biomimetic ligand presentation, and associated cellular responses at the cell-material interface. Understanding how each of these design parameters affects cell response will contribute significantly to the rational engineering of bioactive materials. Potential applications for polymer blends with enhanced mechanical and biological properties include surface coatings on vascular grafts and stents, as well as composite materials for tissue engineered scaffolds and vascular substitutes.

Thesis (M. S.)--Mechanical Engineering, Georgia Institute of Technology, 2009.
Committee Chair: Nerem, Robert M.; Committee Member: Gleason, Rudolf L.; Committee Member: Taylor, W. Robert; Committee Member: Vito, Raymond P.; Committee Member: Wang, Yadong.

In several cases, current therapies available to treat a large number of musculoskeletal system diseases are unsatisfactory as they provide only temporary or partial restoration of the damaged or degenerated site. In an attempt to maintain a high standard of life quality and minimise the economic losses due to the treatments of these frequently occurring ailments and subsequent lost working days, alternative therapies are being explored. Contrary to the current treatments, tissue engineering aims to regenerate the impaired tissue rather than repair and alleviate the symptoms; thus offering a definitive solution. The annulus fibrosus (AF) of the intervertebral disc (IVD) is a musculoskeletal system component frequently subjected to degeneration and rupture, characterised by predominance of anisotropically arranged collagen fibres. In the present thesis, electrospinning technology is used to fabricate polycaprolactone (PCL) scaffolds intended to replicate the anisotropic structure of the AF.

Background: Decellularization of tendon tissue plays a pivotal role in current tissue engineering approaches for in vitro research as well as for translation of graft-based tendon restoration into clinics. Automation of essential decellularization steps like freeze-thawing is crucial for the development of more standardized decellularization protocols and commercial graft production under good manufacturing practice (GMP) conditions in the future. Methods: In this study, a liquid nitrogen-based controlled rate freezer was utilized for automation of repeated freeze-thawing for decellularization of equine superficial digital flexor tendons. Additional tendon specimens underwent manually performed freeze-thaw cycles based on an established procedure. Tendon decellularization was completed by using non-ionic detergent treatment (Triton X-100). Effectiveness of decellularization was assessed by residual nuclei count and calculation of DNA content. Cytocompatibility was evaluated by culturing allogeneic adipose tissue-derived mesenchymal stromal cells on the tendon scaffolds. Results: There were no significant differences in decellularization effectiveness between samples decellularized by the automated freeze-thaw procedure and samples that underwent manual freeze-thaw cycles. Further, we inferred no significant differences in the effectiveness of decellularization between two different cooling and heating rates applied in the automated freeze-thaw process. Both the automated protocols and the manually performed protocol resulted in roughly 2% residual nuclei and 13% residual DNA content. Successful cell culture was achieved with samples decellularized by automated freeze-thawing as well as with tendon samples decellularized by manually performed freeze-thaw cycles. Conclusions: Automated freeze-thaw cycles performed by using a liquid nitrogen-based controlled rate freezer were as effective as previously described manual freeze-thaw procedures for decellularization of equine superficial digital flexor tendons. The automation of this key procedure in decellularization of large tendon samples is an important step towards the processing of large sample quantities under standardized conditions. Furthermore, with a view to the production of commercially available tendon graft-based materials for application in human and veterinary medicine, the automation of key procedural steps is highly required to develop manufacturing processes under GMP conditions.

Bioengineering
Ph.D.
60 % of school children have some form of untreated tooth decay or have suffered trauma to the front teeth which results in pulp damage. If left untreated, these teeth are susceptible to premature fracture/loss under daily stresses. In cases of adolescent tooth loss, teenagers cannot get dental implants until after the growth spurts; their only option is using removable dentures which lowers their quality of life. Conventional endodontic treatment (root canal treatment) is used in cases of pulp necrosis, but cannot be performed in immature permanent teeth due to major differences in tooth anatomy. Currently the American Dental Academy has approved a procedure called Regenerative Endodontic Treatment (RET) for such cases, but the outcomes are still unpredictable and the method is largely unreliable. One issue that we are trying to address in this work is the regeneration of the pulp-dentin complex (PDC), specifically the interface. Endeavors in regenerating either pulp or dentin have been successful individually, but the interface region is the anatomical and physiologic hallmark of the PDC and has not been addressed. We have proposed a biomimetic scaffold to facilitate early stage stratification of these different tissues and allow recapitulation of their interface. Tissue engineering principles and biomaterial processing techniques were used simultaneously to encourage dental pulp stem cells into mineralize selectively only on one side. This effectively allows the scaffold to serve as the interface region between the hard dentin and the soft vascular pulp.
Temple University--Theses

In this thesis, the suitability of Melt Electrospinning Writing technology is demonstrated for two applications: building functional tissue substitutes and engineering relevant models to study disease mechanisms. More specifically, a myocardial patch was built for cardiac tissue engineering, and a prostate microtissue was engineered to study the interactions between epithelium and stroma during prostate cancer progression. This thesis corresponds to the dual Master in Biofabrication, carried out between Universiteit Utrecht and Queensland University of Technology.

Tissue engineering and regenerative medicine aim to develop materials that mimic some of the characteristics of the tissue they are replacing and control the growth and proliferation of cells. Despite exceptional advances in the range and quality of materials used, much remains to discover about the processes regulating interfaces between cells and their surroundings, or at cell-material interfaces. In order to study and control such interactions, scientists have produced engineered matrices aiming to mimic some of the feature of natural extra-cellular matrix (biochemistry, geometry/topography and mechanical properties). In order to pattern 2D-nanofibers on relatively large areas and throughput, allowing comprehensive biological studies, we developed a nano-fabrication technique based on the deposition of sparse mats of electrospun fibres with different diameters. These mats are used as masks to grow cell resistant polymer brushes from exposed areas. After removal of the fibres, the remaining brushes define a quasi-2D fibrous pattern onto which ECM molecules such as fibronectin can be adsorbed. Chapter 2 includes details of the techniques used to produce and characterize the fibrous nanopattern. Chapter 3 is focused on cell phenotype observed on the different nanofibres sizes. Adhesion assays showed that cell spreading, shape and polarity are regulated by the size of fibres but also the density of the nanofibres, similarly to previous observations made on circular nanopatterns. We then focused on the study of focal adhesion formation and maturations on these nanofibres and the role of key proteins involved in the regulation of the adhesion plaque: integrins and vinculin. Cells expressing different integrins were found to sense the nanoscale geometry differently. Vinculin sensing is the topic of Chapter 4. Although vinculin recruitment dynamics was affected by the nanofibrous patterns and focal adhesions arrange differently on the nanofibres, this protein does not seem to mediate nanoscale sensing. In Chapter 5, we finally focused on the role of the actin cytoskeleton as a direct sensor of nanoscale geometry. A gradual decrease in stress fibre formation was observed as the nanofibres dimensions decrease. Live imaging also demonstrated that the geometry of the extracellular environment strongly affects cytoskeleton rearrangement, stress fibres formation and disassembly. We identify the role of cytoskeleton contractility as an important sensor of the nanoscale geometry. Our study provides a deeper insight in understanding cell adhesion to the extracellular environment and the role of the matrix geometry and topography on such phenomena, but also raises questions regarding the more detailed molecular sensory elements enabling the direct sensing of nanoscale geometry through the actin cytoskeleton.

Thesis (M. S.)--Biomedical Engineering, Georgia Institute of Technology, 2009.
Committee Chair: Wang, Yadong; Committee Member: Bao, Gang; Committee Member: Bellamkonda, Ravi; Committee Member: Boyan, Barbara; Committee Member: Chaikof, Elliot; Committee Member: Meredith, J. Carson.

Tissue engineering has emerged as a promising alternative to conventional orthopaedic grafting therapies. The general paradigm for this approach, in which phenotype-specific cells and/or bioactive growth factors are integrated into polymeric matrices, has been successfully applied in recent years toward the development of bone, ligament, and cartilage tissues in vitro and in vivo. Despite these advances, an optimal cell source for skeletal tissue repair and regeneration has not been identified. Furthermore, the lack of robust, functional orthopaedic tissue interfaces, such as the bone-ligament enthesis, severely limits the integration and biological performance of engineered tissue substitutes. This works aims to address these limitations by spatially controlling the genetic modification and differentiation of fibroblasts into a mineralizing osteoblastic phenotype within three-dimensional polymeric matrices. The overall objective of this project was to investigate transcription factor-based gene therapy strategies for the differentiation of fibroblasts into a mineralizing cell source for orthopaedic tissue engineering applications. Our central hypothesis was that fibroblasts genetically engineered to express Runx2 via conventional and biomaterial-mediated ex vivo gene transfer approaches will differentiate into a mineralizing osteoblastic phenotype. We have demonstrated that a combination of retroviral Runx2 overexpression and glucocorticoid hormone treatment synergistically induces osteoblastic differentiation and biological mineral deposition in primary dermal fibroblasts cultured in monolayer. We report for the first time that glucocorticoids induce osteoblastic differentiation in this model system by modulating the phosphorylation state of a negative regulatory serine residue (Ser125) on Runx2 through an MKP-1-dependent mechanism. Furthermore, we utilized these Runx2-genetically engineered fibroblasts to create mineralized templates for bone repair in vitro and in vivo. Finally, we engineered a heterogeneous bone-soft tissue interface with a novel biomaterial-mediated gene transfer approach. Overall, these results are significant toward the ultimate goal of regenerating complex, higher-order orthopaedic grafting templates which mimic the cellular and microstructural characteristics of native tissue. Cellular therapies based on primary dermal fibroblasts would be particularly beneficial for patients with a compromised ability to recruit progenitors to the sight of injury as result of traumatic injury, radiation treatment, or osteodegenerative disease.

Bone defects caused by trauma, tumor resection or disease present a significant clinical problem. Failures in 'high risk' fractures and large bone defects have been reported to be as high as 30-50%. The drawbacks associated with current bone grafting procedures have stimulated the search for improved techniques for bone repair. Tissue engineering/regenerative medicine approaches promote tissue repair by providing a combination of physical and biological cues through structural scaffolds and bioactive agents. Though they have demonstrated significant promise for bone regeneration, very little has been translated to clinical practice. The goal of this thesis was to investigate the potential of electrospun nanofiber mesh scaffolds for bone regeneration. Nanofiber meshes were utilized in a three-pronged approach. First, we validated their ability to robustly support osteogenic cell functions, including proliferation and matrix mineralization. We also demonstrated their efficacy as a cell delivery vehicle. Second, we investigated the effects of modulating nanofiber bioactivity and orientation on stem cell programming. Our results indicate that functionalization of nanofiber meshes with a collagen-mimetic peptide enhanced the migration, proliferation and osteogenic differentiation of cells. Fiber alignment improved cell migration along the direction of fiber orientation. Finally, a nanofiber mesh based hybrid system for growth factor delivery was developed for bone repair and tested in a challenging animal model. The delivery of bone morphogenetic protein (BMP) via this system resulted in the functional restoration of limb function, and in fact proved more efficacious than the current clinical standard for BMP delivery. The studies performed in this thesis have suggested novel techniques for improving the repair of clinically challenging bone defects. They indicate that the delivery of BMP via the hybrid system may reduce the dose and side effects of BMP, thereby broadening the use of BMP based bone augmentation procedures. Therefore, this nanofiber mesh based system has the potential to become the standard of care for clinically challenging bone defects, including large bone defects, open tibial fractures, and nonunions.

Skeletal muscle is an essential tissue for several vital functions. It displays an intrinsic regenerative ability in case of injury, thanks to the activation of satellite cells (SCs), the adult skeletal muscle stem cells. In presence of large defects, the renewing capacities of skeletal muscle are compromised. In such situations regenerative medicine may be a promising solution. This project is focused on a neonatal pathology known as congenital diaphragmatic hernia (CDH), in which the diaphragm fails to close during gestation. CDH is a severe anomaly with an incidence of 1 on 2,500-3,000 new-borns and high mortality rate. Currently, the most frequently used material for the surgical CDH repair is polytetrafluoroethylene (Gore-Tex[R]), but its application can lead to several drawbacks, as hernia recurrence and chest deformation. Great interest has been shown in alternative solutions based on tissue engineering approaches. In this regard, the use of decellularised extracellular matrix (ECM) revealed to be encouraging. When transplanted in vivo it can integrate with the native tissue, recruit host stem cells and influence their behaviour towards a regenerative process. The aim of this work is to characterise a novel tissue engineering approach based on the use of diaphragm decellularised ECM (dECM) as an alternative solution to the current CDH clinical options. The final purpose is to close the defect on the diaphragm and to induce its regeneration and functional recovery. In vivo, we created the first surgical CDH mouse model and we repaired the defect on the diaphragm using mouse dECM and expanded-polytetrafluoroethylene (ePTFE) as control. The transplantation of dECM patches did not cause any rejection effect nor hernia recurrence, differently from ePTFE treated mice. Moreover, ePTFE patches induced a foreign body reaction that was absent when dECM patches were used. We further considered three essential aspects of tissue regeneration: new muscle tissue formation, angiogenesis and re-innervation. In all the cases the biologic patch demonstrated to be better compared to ePTFE. The prolonged activation of muscle regeneration together with the angiogenic and re-innervation processes induced by dECM translated into an overall amelioration of diaphragmatic function compared to ePTFE-treated animals. Despite the positive clinical outcome, dECM patches did not activate complete regeneration of the defect. For this reason, we set up a tissue engineering technique to re-create in vitro diaphragmatic muscle tissues recellularising mouse diaphragm dECM and human MPCs cells. The aim was to obtain skeletal muscle-like substitutes for CDH capable to boost myofibers generation and further improve tissue functionality. We demonstrated that human MPCs not only were able to engraft the scaffold and repopulate the dECM in all its thickness, but most importantly, they differentiated giving rise to metabolic active myotubes. Moreover, a subpopulation of cells maintained SCs features, showing the ability to respond to in vitro injury. Given the positive outcomes obtained using dECM, the next step to get closer to clinic would be to use larger animal models. Moreover, the recellularisation could be improved by using mechanical stimulation, perfusion systems and by adding other cell types as endothelial and neural cells, in order to obtain a more complete in vitro construct for pre-clinical and clinical applications. Finally, the two parts of this project could be joined by closing the defect on the diaphragm using recellularised ECM, with the aim to favour tissue regeneration and reduce the drawbacks related to the use of current synthetic patches.
Il muscolo scheletrico ha un’intrinseca capacità rigenerativa grazie all’attività svolta dalle cellule satelliti. In presenza di danni estesi però tali capacità rigenerative possono essere compromesse. In queste situazioni un approccio di medicina rigenerativa può costituire una soluzione promettente. Questo progetto è focalizzato sull’ernia diaframmatica congenita, patologia neonatale caratterizzata da un’incompleta formazione del diaframma, con incidenza di 1 su 2,500-3,000 neonati e un alto tasso di mortalità. Attualmente, il materiale più usato per il riparo dell’ernia è il politetrafluoroetilene (Gore-Tex[R]), tuttavia il suo utilizzo può causare effetti collaterali, come la ricorrenza dell’ernia e malformazioni della cassa toracica. Grande interesse è stato rivolto a soluzioni di ingegneria tissutale, come l’uso di matrici extracellulari decellularizzate. Quando trapiantate in vivo esse riescono ad integrarsi in maniera fisiologica con il tessuto nativo e reclutano cellule staminali, modulando il loro comportamento verso un processo rigenerativo. Lo scopo di questo progetto è caratterizzare un approccio di ingegneria tissutale basato sull’uso di matrici decellularizzate come soluzione alternativa all’attuale metodo per il riparo l’ernia. L’obiettivo è chiudere il difetto sul diaframma ed indurne la rigenerazione. In vivo, abbiamo creato il primo modello murino di ernia diaframmatica e abbiamo riparato il difetto usando una matrice decellularizzata. Il politetrafluoroetilene espanso (ePTFE) è stato usato come controllo. Il trapianto di matrici decellularizzate non ha causato rigetto o ricorrenza dell’ernia, a differenza degli animali trattati con ePTFE. Inoltre, ePTFE ha indotto una reazione da corpo estraneo che era completamente assente negli animali trattati con la matrice biologica. Ci siamo poi concentrati su tre aspetti fondamentali della rigenerazione: la formazione di nuovo tessuto muscolare, angiogenesi e re-innervazione. In tutti i casi la matrice biologica ha dimostrato di essere migliore di quella sintetica. La prolungata attivazione della rigenerazione muscolare insieme ai processi angiogenici e di re-innervazione indotti dalla matrice extracellulare si sono tradotti in un generale miglioramento delle funzioni diaframmatiche rispetto a quanto ottenuto negli animali con ePTFE. Nonostante i risultati positivi, la matrice extracellulare non era in grado di indurre una completa rigenerazione del difetto. Perciò abbiamo messo a punto una tecnica di ingegneria tissutale per ricreare in vitro tessuti diaframmatici ricellularizzando matrici decellularizzate con precursori muscolari umani. Lo scopo era di ottenere dei possibili costrutti paragonabili al muscolo scheletrico da usare per il riapro dell’ernia in modo da stimolare maggiormente la generazione di nuove miofibre e migliorare la funzionalità tissutale. I precursori muscolari umani erano in grado di attecchire sulla matrice decellularizzata, di ripopolarla in tutto il suo spessore e di differenziare dando origine a miotubi attivi metabolicamente. Inoltre, una sottopopolazione di cellule manteneva le caratteristiche tipiche delle cellule satelliti, dimostrando di saper rispondere in vitro ad un danno. Visti i risultati positivi ottenuti usando la matrice decellularizzata, il passaggio successivo per avvicinarsi alla cinica è rappresentato dall’utilizzo di modelli animali più grandi. Inoltre, la ricellularizzazione potrebbe essere migliorata grazie a stimolazione meccanica, a sistemi di perfusione e all’aggiunta di altri tipi cellulari (cellule endoteliali e neurali) con lo scopo di ottenere un costrutto più completo per possibili applicazioni pre-cliniche e cliniche. Infine, le due parti di questo progetto potrebbero essere unite in futuro riparando il difetto sul diaframma usando matrici biologiche ricellularizzate al fine di favorire la rigenerazione e ridurre gli svantaggi legati all’uso delle matrici sintetiche.

Pre-clinical studies are a necessary step in the process of material and drug testing. For this, high-throughput monolayer cell cultures are conducted followed by in vivo animal experiments. However, animal use is ethically questionable and in many cases yields misleading results. In vitro three dimensional (3D) tissue engineered (TE) structures have been shown to better represent in vivo tissue morphology and biochemical pathways than monolayer cultures and are less ethically questionable than animal models. Therefore, an in vitro biomimetic musculoskeletal junction (MSKjct) is required as a more relevant pre-clinical testbed. This thesis describes the steps taken to co-culture 3D TE skeletal muscle and bone models as a material testbed and towards an in vitro MSKjct.

The deposition of cells at sites of injury is a clinically relevant approach to facilitate local tissue regeneration and repair. However, cell engraftment, retention, and survival are generally modest, requiring the development of novel deposition techniques and biomaterials. Here, a micro-sized polymeric network (microMESH) is investigated as a promising biodegradable scaffold for the engraftment and tissue integration of human Adipose-Derived Stem Cells (hADSCs) to be used for a wide range of injuries, including myocardial infarction. microMESH comprises a regular network of PLGA microfilaments spatially organized to form square openings of 5x5, 10x10 and 20x20 μm2 . microMESH is realized using soft lithographic techniques starting from a master silicon template reproducing the actual geometry of the final PLGA network. After extensive geometrical, physico-chemical, and mechanical characterizations using a broad range of techniques, hADSCs were integrated with microMESH. Cell viability, spatial organization, secretome and stemness were characterized for all three different microMESH configurations and compared to conventional systems, including 2D plastic dishes and collagen layers. Interestingly, when hADSCs were cultured on microMESH they organized in spheroidal-like structures, despite the geometry, maintaining viability over time. This peculiar attitude of the microMESH to form assemblies better represents the human tissue outside the body, compared to 2D monolayer cultures. Additionally, spheroids established an intimate interaction with the microMESH resulting in the scaffold incorporation within the 3D arrangement formed by the cells. On the contrary, hADSCs form only superficial interaction above the flat collagen sheet that is currently used for cell transplantation in animal models of cardiac diseases. Moreover, once the hADSCs are placed on microMESH, the actin cytoskeleton reorganizes to confer a 3D cell shape with multidirectional actin arrangements, forming nonlinear structures and ring structures at the anchorage site to the microMESH, relative to linear filaments when the cells adhered and flattened onto the plastic surface and on top of collagen scaffold. This internal reorganization and the stronger interaction may explain why microMESH scaffold fostered the secretion of biologically active molecules, acting in a paracrine fashion on resident cells, which are expected to accelerate tissue regeneration and repair. Specifically, when hADSCs grew on microMESH we observed a trend for higher production of several factors with specific implications in angiogenesis, stem cell proliferation and expansion, cell survival, inflammation modulation, ECM remodeling, stem cell mobilization, chemotaxis and homing, relative to 2D monolayer conditions. The paracrine effect of hADSCs is scaffold dependent and can be modulated by tailoring the geometrical and mechanical properties of microMESH. Indeed, the 5x5 microMESH showed its contribution in angiogenesis, ECM remodeling and stem cell mobilization from bone marrow into the bloodstream. Indeed, highest amounts of VEGF, TIMP-2 and GCSF, respectively were detected in 5x5 geometry compared to the other conditions. Rather, 10x10 geometry promotes angiogenesis enhancing the VEGF production, stem cell proliferation and survival by raising the Fibroblast Growth Factors family secretion and EGF factor, respectively and favors ECM remodeling increasing the TIMP-2 production compared to other conditions. Lastly, the 20x20 seems to have a more anti-inflammatory role (combination of IL-10 and TGF-β1) and chemotactic function (e.g. RANTES). Finally, in this work, we started to shed new light on the ability of micromMESH geometry to modulate the hADSCs stemness evaluating the expression levels of CD44, CD90 and CD105 markers over time. The proposed microMESH scaffold is expected to provide an effective alternative to more conventional hADSCs transplant techniques.