Current therapy for chronic kidney disease (CKD) is limited; however, recent studies have shown that a subpopulation of cells derived from the bone marrow, known as early outgrowth cells (EOCs), are able to attenuate kidney injury. Here we examined the efficacy of a modular tissue engineering system whereby the EOCs might be easily removed in the event of malignant change. While modular therapy mimicked the effects seen with standard EOC therapy, the modules degraded allowing the encapsulated EOCs to enter systemic circulation. Given the presumed egress of EOCs, we explored an alternative strategy for kidney protection. Here we investigated the long-term effectiveness of administering the conditioned medium (EOC-CM) that contains the factors the EOCs secrete, rather than the cells themselves. In these studies, repeated administration of EOC-CM attenuated the structural and functional manifestations of kidney injury suggesting that this approach may provide an effective and feasible, cell-free approach for CKD.

Despite the increasing clinical demand in reconstructive, cosmetic and correctional surgery there remains no optimal strategy for the regeneration or replacement of adipose tissue. Previous approaches to adipose tissue engineering have failed to create an adipose tissue depot that maintains implant volume in vivo long-term (>3 months). This is due to inadequate mechanical properties of the biomaterial and insufficient vascularization upon implantation. Modular tissue engineering is a means to produce large volume functional tissues from small sub-mm sized tissues with an intrinsic vascularization. We first explored the potential of a semi-synthetic collagen/poloxamine hydrogel with improved mechanical properties to be used as the module biomaterial. We found this biomaterial to not be suitable for adipose tissue engineering because it did not support embedded adipose-derived stem cell (ASC) viability, differentiation and human microvascular endothelial cell (HMEC) attachment. ASC-embedded collagen gel modules coated with HMEC were then implanted subcutaneously in SCID mice to study its revascularization potential. ASC cotransplantation was shown to drive HMEC vascularization in vivo: HMEC were seen to detach from the surface of the modules to form vessels containing erythrocytes as early as day 3; vessels decreased in number but increased in size over 14 days; and persisted for up to 3 months. Early vascularization promoted fat development. Only in the case of ASC-HMEC cotransplantation was progressive fat accumulation observed in the module implants. Although implant volume was not maintained, likely due rapid collagen degradation, the key result here is that ASC-HMEC cotransplantation in the modular approach was successful in creating vascularized adipose tissue in vivo that persisted for 3 months. The modular system was then studied in vitro to further understand ASC-EC interaction. Coculture with ASC was shown to promote an angiogenic phenotype (e.g. sprouting, migration) from HUVEC on modules. RT-PCR analysis revealed that VEGF, PAI-1 and TNFα was involved in ASC-EC paracrine signalling. In summary, ASC-HMEC cotransplantation in modules was effective in rapidly forming a vascular network that supported fat development. Future work should focus on further elucidating ASC-EC interactions and developing a suitable biomaterial to improve adipose tissue development and volume maintenance of engineered constructs.

Current therapy for chronic kidney disease (CKD) is limited; however, recent studies have shown that a subpopulation of cells derived from the bone marrow, known as early outgrowth cells (EOCs), are able to attenuate kidney injury. Here we examined the efficacy of a modular tissue engineering system whereby the EOCs might be easily removed in the event of malignant change. While modular therapy mimicked the effects seen with standard EOC therapy, the modules degraded allowing the encapsulated EOCs to enter systemic circulation. Given the presumed egress of EOCs, we explored an alternative strategy for kidney protection. Here we investigated the long-term effectiveness of administering the conditioned medium (EOC-CM) that contains the factors the EOCs secrete, rather than the cells themselves. In these studies, repeated administration of EOC-CM attenuated the structural and functional manifestations of kidney injury suggesting that this approach may provide an effective and feasible, cell-free approach for CKD.

<p>Massive loss of cardiac tissue as a result of myocardial infarction can create a poorly-conducting substrate with impaired contractility, ultimately leading to heart failure and lethal arrhythmias. Recent advances in pluripotent stem cell research have provided investigators with potent sources of cardiogenic cells that may be transplanted into failing hearts to provide electrical and mechanical support. Experiments in both small and large animal models have shown that standard cell delivery techniques suffer from poor retention and engraftment of cells. In contrast, the transplantation of engineered cardiac tissues may provide improved cell retention at the injury site, creating a more localized paracrine effect and yielding more efficient structural and functional repair. However, tissue engineering methodologies to assemble cardiomyocytes or cardiac progenitors into aligned, 3-dimensional (3D) myocardial tissues capable of physiologically relevant electrical conduction and force generation are lacking. The objective of this thesis was thus to develop a methodology to generate highly functional engineered cardiac tissues starting from pluripotent stem cells.</p><p>To accomplish this goal, we first derived purified populations of cardiac myocytes from mouse embryonic stem cells (mESC-CMs) by antibiotic selection driven by an &#945;-myosin heavy-chain promoter. Culture conditions that yielded robust mESC-CM electrical coupling and fast action potential propagation were optimized in confluent cell monolayers. We then developed a microfabrication-based tissue engineering approach to create engineered cardiac tissues ("patches") with uniform 3D cell alignment. We found that, unlike in monolayers, mESC-CMs required a population of supporting cardiac fibroblasts to enable the formation of 3D engineered tissues. Detailed structural, electrical and mechanical characterization demonstrated that engineered cardiac patches consisted of dense, uniformly aligned, highly differentiated and electromechanically coupled mESC-CMs and supported rapid action potential conduction velocities between 22 - 25cm/s and contractile force amplitudes of up to 2mN. </p><p>Next, we sought to circumvent the use of primary cardiac fibroblasts by utilizing a single pluripotent stem cell-derived source, multipotent cardiovascular progenitors (CVPs) capable of differentiating into vascular smooth muscle and endothelial cells in addition to cardiomyocytes. CVPs were derived from mouse embryonic stem cells and induced pluripotent stem (iPS) cells by antibiotic selection driven by an Nkx2-5 enhancer element. Similar to mESC-CMs, CVPs formed highly differentiated cell monolayers with electrophysiological properties that improved with time in culture to levels achieved with pure mESC-CMs. However, unlike mESC-CMs, CVPs formed highly functional 3D engineered cardiac tissues without the addition of cardiac fibroblasts, enabling engineered cardiac tissues to be formed from a single, entirely stem cell-derived source.</p><p>Finally, we explored mechanisms of synergistic cardiac fibroblast/myocyte signaling in 3D engineered tissues by using cardiac fibroblasts of different developmental stages in the settings of direct 3D co-culture as well as in conditioned media studies. When co-cultured with fetal cardiac fibroblasts, mESC-CMs were capable of two-fold faster action potential propagation and 1.5-fold higher maximum contractile force generation than when co-cultured with adult cardiac fibroblasts. These functional improvements were associated with enhanced mESC-CM spreading and upregulation of important ion channel, coupling, and contractile proteins. Conditioned medium studies revealed that compared to adult fibroblasts, fetal cardiac fibroblasts secreted distinct paracrine factors that promoted mESC-CM spreading and spontaneous contractility in 3D engineered tissues and acted via the MEK-ERK pathway. Quantitative gene expression analysis revealed paracrine factor candidates that may mediate this action.</p><p>In summary, this thesis presents methods and underlying mechanisms for generation of highly functional cardiac tissues from pluripotent stem cell sources. These techniques and findings provide foundation for future engineering of human ES and iPS cell-based cardiac tissues for therapeutic and drug screening applications.</p> / Dissertation

Growth plate is a highly organized cartilaginous tissue found at the end of long bones and is responsible for longitudinal growth of the bones. Growth plate fracture leads to retarded growth and unequal limb length, which might have a lifelong effect on a person’s physical stature. This research is a tissue engineering approach for the treatment of growth plate injury. Insulin-like growth factor I (IGF-I), which can stimulate cartilage formation, was encapsulated within PLGA microspheres that were then used to form porous scaffolds. The release profile of the IGF-I from the PLGA scaffold showed a biphasic release pattern. In vitro studies were done by seeding rat bone marrow cells (BMCs) on the top of IGF-I encapsulated PLGA scaffolds, and the results showed an increase in cell multiplication and glycosaminoglycan content. The final in vivo studies were conducted by creating growth plate injury and implanting scaffolds in the tibiae of the New-Zealand white rabbits. Histological analysis of tissue sections showed regeneration of cartilage, albeit with disorganized structure, at the site of implantation of IGF-I encapsulated scaffolds. This work will be a significant step towards tissue engineering of growth plate cartilage.

Repair of bone defects is a major challenge in orthopaedic surgery. Current bone graft treatments, including autografts, allografts and xenografts, have many limitations making it necessary to develop a biomaterial to be a bone graft substitute. One such biomaterial is bioactive resorbable silica-calcium phosphate nanocomposite (SCPC). SCPC was processed using a 3D rapid prototyping technique and sintered at different temperatures to create porous scaffolds. SEM analyses and mercury intrusion porosimetry showed SCPC to be highly porous with micro- and nanopores. BET analysis indicated that SCPC had high surface area. Mechanical testing demonstrated that SCPC had a compressive strength similar to trabecular bone. Analysis of different thermal treatment temperatures indicated as the temperature was increased, the porosity decreased and the mechanical strength increased. When loaded with rhBMP-2 (SCPC-rhBMP-2), SCPC provided a sustained release profile of rhBMP-2 for 14 days. This was shown to be a greater release than hydroxyapatite (HA)-rhBMP-2. After immersion in SBF, ICP analyses showed the calcium concentration of SBF dropped drastically after one day of immersion. In conjunction, FTIR showed the formation of a hydroxyapatite layer on the SCPC surface and was confirmed by SEM. SCPC thermally treated at 850 ??C demonstrated the greatest dissolution/precipitation reactions when immersed in SBF. Processing the SCPC-rhBMP-2 hybrid using a rapid prototyping technique allowed for an exact replica of the rabbit ulna to be fabricated. This was implanted into a 10 mm segmental defect in the rabbit ulna. CT scans during the healing of the defect showed intimate union between SCPC-rhBMP-2 and the bone and about 65% healing of the defect after 4 weeks. Rabbits were euthanized after 12 and 16 weeks. Digital images show almost complete healing of the defect after 16 weeks. Torsional testing of the ulna after 12 weeks demonstrated restoration of maximum torque and angle at failure. Histological evaluation after 12 weeks showed the regenerated bone has all the morphological characteristics of mature bone. Through in-vitro and in-vivo testing, it can be recommended that the porous bioactive SCPC can serve as a successful delivery system for biological growth factors and serve as an alternative to autologous bone grafting.

Hypercholesterolemia is a risk factor for osteoporosis but the underlying mechanism is unknown. Previous evidence suggests that osteoporosis results from an impaired regulation of osteoblasts by fluid pressure fluctuations in the bone matrix. Recently, our laboratory showed that enhanced cholesterol in the cell membrane, due to hypercholesterolemia, alters leukocyte mechanosensitivity. We predict a similar link between osteoblasts and hypercholesterolemia leading to osteoporosis. Specifically, we hypothesize that extracellular cholesterol modifies the osteoblast sensitivity to pressure. MC3T3-E1 cells were exposed to hydrodynamic pressures regimes (mean=40mmHg, amplitude=0-20mmHg, frequency=1Hz) for 1-12 hours. To assess the impact of membrane cholesterol enrichment, cells were pre-treated with 0-50 µg/mL cyclodextran:cholesterol conjugates. We assessed the pressure effects on mitosis and F-actin stress fiber formation (SFF) of cells. Exposure of cells to 50/30 mmHg pressure transiently increased the number of cells in the S- and G2M-phases of mitosis after 6 and 12 hours, respectively. Relative to controls, osteoblast-like cells exposed to all pressures exhibited significantly (p

<p><p> Articular cartilage is a smooth connective tissue that covers the ends of bones and protects joints from wear. Cartilage has a poor healing capacity, and the lack of treatment options motivates the development of tissue engineering strategies. The widespread cartilage degeneration associated with osteoarthritis (OA) is dramatically accelerated by joint injury, but the defined initiating event presents a therapeutic window for preventive treatments. In vitro model systems allow investigation of OA risk factors and screening of potential therapeutics. This dissertation develops stem-cell based strategies to 1) treat cartilage injury and OA using tissue-engineered cartilage, 2) prevent the development of OA by delivering stem cells to the joint after injury, and 3) study cartilage by establishing systems to model genetic and environmental contributors to OA.</p><p> Adipose-derived stem cells (ASCs) and bone marrow-derived mesenchymal stem cells (MSCs) are promising human adult cell sources for cartilage tissue engineering, but require distinct chondrogenic conditions. As compared to ASCs, MSCs demonstrated enhanced chondrogenesis in both alginate beads and cartilage-derived matrix scaffolds. </p><p> We hypothesized that MSC therapy would prevent post-traumatic arthritis (PTA) by altering the balance of inflammation and regeneration. Highly purified MSCs (CD45-TER119-PDGFR&#945;+Sca-1+) rapidly expanded under hypoxic conditions. Unexpectedly, MSCs from control C57BL/6 (B6) mice proliferated and differentiated more than MSCs from MRL/MpJ (MRL) "superhealer" mice. We injected B6 or MRL MSCs into mouse knees immediately after fracture, and MSCs of either strain were sufficient to prevent PTA. </p><p> Genetically reprogramming adult cells into induced pluripotent stem cells (iPSCs) generates large numbers of patient-matched cells with chondrogenic potential for therapy and cartilage modeling. We produced murine iPSC-derived cartilage constructs with a multi-phase approach involving micromass culture with bone morphogenetic protein-4, flow cytometry cell sorting of chondrocyte-like cells, monolayer expansion, and pellet culture with transforming growth factor-beta 3. Successful differentiation was confirmed by increased chondrogenic gene expression, robust synthesis of glycosaminoglycans and type II collagen, and the repair of an in vitro cartilage defect. </p><p> The diverse applications pursued in this research illustrate the power of stem cells to deepen the understanding of cartilage and guide the development of therapies to prevent and treat cartilage injury and OA.</p> / Dissertation

<p>The aim of this dissertation was to develop new techniques for producing electrospun scaffolds for use in the tissue engineering of articular cartilage. We developed a novel method of imparting mechanical anisotropy to electrospun scaffolds that allowed the production of a single, cohesive scaffold with varying directions of anisotropy in different layers by employing insulating masks to control the electric field. We improved the quantification of fiber alignment, discovering that surface fibers in isotropic scaffolds show similar amounts of fiber alignment as some types of anisotropic scaffolds, and that cells align themselves in response to this subtle fiber alignment. We improved previous methods to improve cellular infiltration into tissue engineering scaffolds. Finally, we produced a new material with chondrogenic potential consisting of native unpurified cartilage which was electrospun as a composite with a synthetic polymer. This work provided advances in three major areas of tissue engineering: scaffold properties, cell-scaffold interaction, and novel materials.</p> / Dissertation

Degenerative disc disease is a common ailment with enormous medical, psychosocial and economic ramifications. This study was designed to investigate the utility of a thiol-modified hyaluronan(TMHA) and elastin-like polypeptide(EP) composite material as a potential tissue engineering scaffold to reconstitute the nucleus pulposus in early degenerative disc disease. TMHA and EP were combined in various concentrations and cross-linked using poly(ethylene glycol)diacrylate. Resulting materials were evaluated biomechanically and biologically. Confined compression testing revealed that the addition of EP to TMHA-based gels resulted in a stiffer construct, but remained an order of magnitude less stiff than native nucleus. The in vitro cell culture experiments with human intervertebral disc cells demonstrated 70% cell viability at three weeks with apparent maintenance of phenotype. The addition of EP did not have a significant biologic effect. An in vivo pilot study demonstrated biocompatibility of the TMHA-based hydrogels; additional power is required to adequately assess treatment effect.