Thesis (M.S.)--California Polytechnic State University, 2008. / "June 2008." "In partial fulfillment of the requirements for the degree [of] Master of Science in Engineering with Specialization in Biomedical Engineering." "Presented to the faculty of California Polytechnic State University--San Luis Obispo." Major professor: Lily Laiho, Ph.D. Includes bibliographical references (leaves 76-79). Also available online and on microfiche (2 sheets).

Thesis (Ph. D.)--Biomedical Engineering, Georgia Institute of Technology, 2007. / Bellamkonda, Ravi, Committee Member ; LaPlaca, Michelle, Committee Chair ; Stein, Donald, Committee Member ; Garca̕, Andrš, Committee Member ; Archer, David, Committee Member ; Borlongan, Cesario, Committee Member.

The synthetic biodegradable polymer poly(lactide-co-glycolide) (PLGA) has been widely explored as substrate biomaterials for controlled drug delivery and tissue engineering. ECM component heparin and bone mineral hydroxyapatite (HA) are attractive biomaterials which can functionalize the PLGA surface to improve cell cell response and to bring in the dual growth factor delivery, because heparin and HA both can improve cell responses and bind with various proteins. To combine the osteoconductivity of HA and the controlled drug release of PLGA microspheres, HA coated PLGA microspheres were developed by a 3 hour rapid HA precipitation on the PLGA microsphere surface. Effects of various fabrication parameters on microsphere and HA coating morphology were evaluated. This core-shell composite worked as a dual drug delivery device and demonstrated better cell cell response than PLGA microspheres without HA coating. Three different methods, including osmogen, extractable porogen and gas-foaming porogen, were evaluated to fabricate porous microspheres as injectable cell scaffolds in the tissue engineering. The gas-foaming method produced covered porous PLGA microspheres, on which a skin layer covered all the surface pores. The skin layer was hydrolysed by NaOH to control the surface porosity. The modified open porous microspheres have large continued surface areas between pores, which provided more continued areas for cell adhesion. The porous microspheres with controllable surface porosity and large surface continuity between pores could be novel injectable cell scaffolds. Heparin was immobilized on the open porous PLGA microspheres by a facile layer-by-layer assemble to combine the advantages of porous structure and the protein binding from heparin. The heparin-coated porous microspheres promoted cell adhesion, spreading, proliferation and osteogenic differentiation. Growth factor-like protein lactoferrin was immobilized on the heparin coated porous microspheres, which further enhanced MG-63 proliferation and osteogenic differentiation. The heparin-coated porous microspheres are promising multi-functional devices for controlled drug delivery and injectable cell delivery.

The thesis aims at investigating the connection between nucleus mechanical characteristics with pluripotency state and differentiation associated with altered cell gene expression levels. The project investigates the deformation characteristics of the cell nucleus during unconfined compression in a 3D cell-seeded agarose constructs. The studies report modification in the mechanical behaviour of the nucleus in different embryonic stem cell phenotypes based on various pluripotent states (naïve or primed states) or following triggering of early differentiation. A multi-scale model is also presented to simulate dynamic details of mechanical perturbation to cells during compression. The first chapter presents a review of the relevant literature to introduce current progress in the related research field and the second chapter describes the general methods used in the thesis including cell culture, agarose construct preparation, construct compression and microscopy recording. The third chapter presents findings of studies involving the application of compression to embryonic stem cells in naïve and primed sate within agarose scaffolds. A range of parameters relating to the relative cell/nucleus morphological modifications are recorded with analysis and discussion. Chapter four presents studies that investigate the early differentiation of embryonic stem cells from either the naïve and primed pluripotency, achieved by altering cell culture condition, and further reveals the nuclear mechanical characteristic changes. The fifth chapter describes a multi-scale model developed to simulating the 3D cell-seeded agarose compression reported in previous chapters. This model is also used to estimate cell mechanical parameters and show accurate deformation detail in different locations within the construct. A final discussion of the thesis is provided in chapter 6 with a plan for future work.

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.

No description available.

A engenharia de tecido ósseo associando células-tronco mesenquimais (CTMs) a biomateriais tem sido proposta como tratamento potencial para o reparo de defeitos ósseos, constituindo uma abordagem nova na área da medicina regenerativa e de amplo interesse para as áreas de cirurgia buco-maxilo-facial e ortopedia. A seleção das CTMs mais adequadas e o método utilizado para carreá-las nos sítios de defeitos ósseos são fatores importantes para o sucesso do tratamento. Como a osteoporose reduz a capacidade de regeneração dos ossos, seria de grande importância que a engenharia do tecido ósseo pudesse ser aplicada com sucesso nessa patologia. Assim, foi avaliado o potencial das CTMs de medula óssea (CTMs-MO) e de tecido adiposo (CTMs-TA) associadas ao arcabouço de vitrocerâmica BioS-2P ou a membrana de P(VDF-TrFE)/BT no reparo de defeitos ósseos em ratas osteoporóticas. A osteoporose foi induzida por ovariectomia e comprovada pela análise microtomográfica dos fêmures. Nas ratas osteoporóticas foram criados defeitos ósseos nas calvárias que foram tratados com implantação de BioS-2P associado à CTMs-MO e CTMs-TA ou com a implantação de membrana de P(VDF-TrFE)/BT combinada com a injeção de CTMs-MO e CTMs-TA. Ao final de 4 semanas, as análises microtomográficas e histológica mostraram que não houve formação óssea nos defeitos sem qualquer tratamento, mas nos defeitos tratados com implantação de BioS-2P ou membrana de P(VDF-TrFE)/BT houve formação óssea independente da presença de CTMs. Apenas os defeitos tratados com membrana de P(VDF-TrFE)/BT e injeção de CTMs-MO apresentaram maior formação óssea, mas não ocorreu a regeneração. / Bone tissue engineering based on the combination of mesenchymal stem cells (MSCs) and biomaterials, has been proposed as a potential treatment for the repair of bone defects, constituting a new approach in the field of regenerative medicine and of interest to the areas of oral and maxillofacial surgery and orthopedics. To select the most suitable MSCs and an efficient method to carry them to the bone defects are the key for the successful treatment. Considering that osteoporosis represents a challenge situation, it would be of the utmost importance that bone tissue engineering could be used in this pathological condition. Thus, the aim of this study was to evaluate the potential of MSCs harvested from bone marrow (MSCs-BM) and from adipose tissue (MSCs-AT) associated to a vitreous scaffold (BioS-2P) or to a membrane of P(VDF-TrFE)/BT in regenerate bone defects created in osteoporotic rats. Osteoporosis was induced by ovariectomy and confirmed by microtomography of the femurs. Defects created in calvaria of osteoporotic rats were implanted with either Bios-2P seeded with MSCs-BM and MSCs-AT or a membrane of P(VDF-TrFE)/BT combined with injection of MSCs-BM and MSCs-AT. After 4 weeks, microtomography and histological analyses showed that there was no bone formation in untreated defects but in those treated with BioS-2P or membrane of P(VDF-TrFE)/BT there was bone formation irrespective of the presence of MSCs. Only defects treated with membrane of P(VDF-TrFE)/BT and MSCs-BM injection resulted in greater bone formation but there was not full bone regeneration.

Thesis (Ph.D.)--University of Alberta, 2010. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Experimental Surgery, Department of Surgery. Title from pdf file main screen (viewed on April 24, 2010). Includes bibliographical references.

A engenharia de tecido ósseo associando células-tronco mesenquimais (CTMs) a biomateriais tem sido proposta como tratamento potencial para o reparo de defeitos ósseos, constituindo uma abordagem nova na área da medicina regenerativa e de amplo interesse para as áreas de cirurgia buco-maxilo-facial e ortopedia. A seleção das CTMs mais adequadas e o método utilizado para carreá-las nos sítios de defeitos ósseos são fatores importantes para o sucesso do tratamento. Como a osteoporose reduz a capacidade de regeneração dos ossos, seria de grande importância que a engenharia do tecido ósseo pudesse ser aplicada com sucesso nessa patologia. Assim, foi avaliado o potencial das CTMs de medula óssea (CTMs-MO) e de tecido adiposo (CTMs-TA) associadas ao arcabouço de vitrocerâmica BioS-2P ou a membrana de P(VDF-TrFE)/BT no reparo de defeitos ósseos em ratas osteoporóticas. A osteoporose foi induzida por ovariectomia e comprovada pela análise microtomográfica dos fêmures. Nas ratas osteoporóticas foram criados defeitos ósseos nas calvárias que foram tratados com implantação de BioS-2P associado à CTMs-MO e CTMs-TA ou com a implantação de membrana de P(VDF-TrFE)/BT combinada com a injeção de CTMs-MO e CTMs-TA. Ao final de 4 semanas, as análises microtomográficas e histológica mostraram que não houve formação óssea nos defeitos sem qualquer tratamento, mas nos defeitos tratados com implantação de BioS-2P ou membrana de P(VDF-TrFE)/BT houve formação óssea independente da presença de CTMs. Apenas os defeitos tratados com membrana de P(VDF-TrFE)/BT e injeção de CTMs-MO apresentaram maior formação óssea, mas não ocorreu a regeneração. / Bone tissue engineering based on the combination of mesenchymal stem cells (MSCs) and biomaterials, has been proposed as a potential treatment for the repair of bone defects, constituting a new approach in the field of regenerative medicine and of interest to the areas of oral and maxillofacial surgery and orthopedics. To select the most suitable MSCs and an efficient method to carry them to the bone defects are the key for the successful treatment. Considering that osteoporosis represents a challenge situation, it would be of the utmost importance that bone tissue engineering could be used in this pathological condition. Thus, the aim of this study was to evaluate the potential of MSCs harvested from bone marrow (MSCs-BM) and from adipose tissue (MSCs-AT) associated to a vitreous scaffold (BioS-2P) or to a membrane of P(VDF-TrFE)/BT in regenerate bone defects created in osteoporotic rats. Osteoporosis was induced by ovariectomy and confirmed by microtomography of the femurs. Defects created in calvaria of osteoporotic rats were implanted with either Bios-2P seeded with MSCs-BM and MSCs-AT or a membrane of P(VDF-TrFE)/BT combined with injection of MSCs-BM and MSCs-AT. After 4 weeks, microtomography and histological analyses showed that there was no bone formation in untreated defects but in those treated with BioS-2P or membrane of P(VDF-TrFE)/BT there was bone formation irrespective of the presence of MSCs. Only defects treated with membrane of P(VDF-TrFE)/BT and MSCs-BM injection resulted in greater bone formation but there was not full bone regeneration.

Articular cartilage is an avascular and flexible connective tissue found in joints. It produces a cushioning effect at the joints and provides low friction to protect the ends of the bones from wear and tear/damage. It has poor repair capacity and any injury can result pain and loss of mobility. One of the common forms of articular cartilage disease which has a huge impact on patient's life is arthritis. Research on cartilage cell/tissue engineering will help patients to improve their physical activity by replacing or treating the diseased/damaged cartilage tissue. Cartilage cell, called chondrocyte is embedded in the matrix (Lacunae) and has round shape in vivo. The in vitro monolayer culture of primary chondrocyte causes morphological change characterized as dedifferentiation. Transforming growth factor-beta (TGF-β), a cytokine superfamily, regulates cell function, including differentiation and proliferation. The effect of TGF-β1, 2, 3, and their manipulated forms in biological regulation of primary chondrocyte was investigated in this work. A novel method was developed to isolate and purify the primary chondrocytes from knee joint of neonate Sprague-Dawley rat, and the effect of some supplementations such as hyaluronic acid and antibiotics were also investigated to provide the most appropriate condition for in vitro culture of chondrocyte cells. Addition of 0.1mg/ml hyaluronic acid in chondrocyte culture media resulted an increase in primary chondrocyte proliferation and helped the cells to maintain chondrocytic morphology. TGF-β1, 2 and 3 caused chondrocytes to obtain fibroblastic phenotype, alongside an increase in apoptosis. The healing process of the wound closure assay of chondrocyte monolayers were slowed down by all three isoforms of TGF-β. All three types of TGF-β negatively affected the strength of chondrocyte adhesion. TGF-β1, 2 and 3 up regulated the expression of collagen type-II, but decreased synthesis of collagen type-I, Chondroitin sulfate glycoprotein, and laminin. They did not show any significant change in production of S-100 protein and fibronectin. TGF-β2, and 3 did not change expression of integrin-β1 (CD29), but TGF-β1 decreased the secretion of this adhesion protein. Manipulated TGF-β showed huge impact on formation of fibroblast like morphology of chondrocytes with chondrocytic phenotype. These isoforms also decreased the expression of laminin, chondroitin sulfate glycoprotein, and collagen type-I, but they increased production of collagen type-II and did not induce synthesis of fibronectin and S-100 protein. In addition, the strength of cell adhesion on solid surface was reduced by manipulated TGF-β. Only manipulated form of TGF-β1 and 2 could increase the proliferation rate. Manipulation of TGF-β did not up regulate the expression of integrin-β1 in planar culture system. The implications of this R&D work are that the manipulation of TGF-β by combination of TGF-β1, 2, and 3 can be utilized in production of superficial zone of cartilage and perichondrium. The collagen, fibronectin and hyaluronic acid could be recruited for the fabrication of a biodegradable scaffold that promotes chondrocyte growth for autologous chondrocyte implantation or for formation of cartilage.