Stem cell differentiation is regulated by the complex interplay of multiple parameters, including adhesive intercellular interactions, cytoskeletal and extracellular matrix remodeling, and gradients of agonists and antagonists that individually and collectively vary as a function of spatial locale and temporal stages of development. Directed differentiation approaches have traditionally focused on the delivery of soluble morphogens and/or the manipulation of culture substrates in two-dimensional, monolayer cultures, with the objective of achieving large yields of homogeneously differentiated cells. However, a more complete understanding of stem cell niche complexity motivates tissue engineering approaches to inform the development of physiologically relevant, biomimetic models of stem cell differentiation. The capacity of pluripotent stem cells to simultaneously differentiate toward multiple tissue-specific cell lineages has prompted the development of new strategies to guide complex, three-dimensional morphogenesis of functional tissue structures. The objective of this project was to characterize the spatiotemporal dynamics of stem cell biophysical characteristics and morphogenesis, to inform the development of ESC culture technologies to present defined and tunable cues within the three-dimensional spheroid microenvironment. The hypothesis was that the biophysical and biochemical cues present within the 3D microenvironment are altered in conjunction with morphogenesis as a function of stem cell differentiation stage. Understanding biochemical and physical tissue morphogenesis, including the relationships between remodeling of cytoskeletal elements and intercellular adhesions, associated developmentally relevant signaling pathways, and the physical properties of the EB structure together elucidate fundamental cellular interactions governing embryonic morphogenesis and cell specification. Together, this project has established a foundation for controlling, characterizing, and systematically perturbing aspects of stem cell microenvironments in order to guide the development of complex, functional tissue structures for regenerative therapies.

Ischemia/reperfusion (I/R) injury causes extensive damage to skeletal muscle, often resulting in prolonged functional deficits. This current study determines the efficacy of controlled release of SDF-1α and IGF-1 by conjugation to biodegradable, polyethylene glycol, (PEG)ylated fibrin gel matrix in skeletal muscle repair of an I/R injury. Male Sprague-Dawley rats underwent a 2-hour tourniquet induced I/R injury on their hind limbs. Twenty-four hours post injury the following treatments were administered: PEGylated fibrin gel (PEG-Fib), SDF-1 conjugated PEGylated fibrin gel (PEG-Fib/SDF-1), or dual protein IGF-1 and SDF-1 conjugated PEGylated fibrin gel (PEG-Fibrin/SDF-1/IGF-1. Following 14 days after injury, functional and histological evaluations were performed. There was no significant difference in maximum tetanic force production recovery between PEG-Fib and PEG-Fib/SDF-1 groups. However, PEG-Fib/SDF-1/IGF-1 group resulted in significant improvement of force production relative to the other treatment groups. The same results were found for specific tension. Histological analysis revealed a greater distribution of small myofibers in the PEG-Fib/SDF-1 group than the PEG-Fib group, while the PEG-Fib/SDF-1/IGF-1 group had the smallest distribution of small fibers and similar to controls (uninjured). There were also a greater number of centrally located nuclei in the PEG-Fib/SDF-1 group than the PEG-Fib group, while the PEG-Fib/SDF-1/IGF-1 group had similar values to controls. Although these results confirm the protective role of exogenous IGF-1, SDF-1 did not have an effect on skeletal muscle repair. / text

Human mesenchymal stem cells (hMSCs) are a promising candidate for cell-based therapies given their therapeutic potential and propensity to grow in vitro. However, to generate the cell numbers required for such applications, robust, reproducible and scalable manufacturing methods need to be developed. To address this challenge, the expansion of hMSCs in a microcarrier-based bioreactor system was investigated. Initial studies performed in T-flask monolayer cultures investigated the effect of key bioprocess parameters such as dissolved oxygen concentration (dO2), the level of medium exchange and the use of serum-free media. 20 % dO2 adversely impacted cell proliferation in comparison to 100 % dO2, whilst FBS-supplemented DMEM was found to be the most consistent and cost-effective cell culture medium despite the advances in serum-free cell culture media. Several microcarriers were screened in 100 mL agitated spinner flasks where Plastic P102-L was selected as the optimal microcarrier for hMSC expansion given the high cell yields obtained, its xeno-free composition and effective harvest capacity. The findings from the initial small-scale studies culminated in the successful expansion of hMSCs on Plastic P102-L microcarriers in a fully equipped 5 L stirred-tank bioreactor (2.5 L working volume), the largest reported volume for hMSC microcarrier culture to date. A maximum cell density of 1.68 x 105 cells/mL was obtained after 9 days in culture; further growth was limited by the low glucose concentration and lack of available surface area. A novel, scalable harvesting method was also developed, allowing for the successful recovery of hMSCs. Importantly, harvested hMSCs retained their immunophenotype, multipotency and ability to proliferate on tissue culture plastic.

Healing large bone defects remains a clinical challenge. While autografts are the gold standard treatment for large bone defects, they are limited by availability and donor site pain. Growth factor treatments such as BMP therapy provide a promising alternative but are expensive and present clinical safety concerns, primarily due to delivery of BMPs at supraphysiological doses. Integrins are ECM receptors which mediate crucial cell functions such as adhesion and differentiation. Therefore, understanding the role of integrins in bone formation and directing desired interactions may enable modulation of host cell functions for therapeutic applications. In this work, beta 1 integrins were deleted in osteolineage cells of transgenic mice at three different stages of differentiation to elucidate their role in bone development. We also engineered bioartificial PEG-based matrices which target the pro-osteogenic alpha 2 beta 1 integrin to promote bone healing. Conditional deletion of beta 1 integrins in osteochondroprogenitor cells under the Twist 2 promoter resulted in severe pre-natal skeletal mineralization defects and embryonic lethality. Targeted deletion of beta 1 integrins in osterix-expressing osteoprogenitors resulted in growth abnormalities, reduced calvarial mineralization, impaired femur development, and tooth defects. However, mice lacking beta 1 integrins in osteocalcin-expressing osteoblasts and osteocytes displayed only a mild skeletal phenotype, indicating that beta 1 integrins play an important role in early skeletal development, but are not required for mature osteoblast function. PEG hydrogels functionalized with the integrin-specific GFOGER ligand enhanced bone regeneration, induced defect bridging in combination with low doses of rhBMP-2 and stimulated improved bone healing compared collagen sponges, which are the clinical standard delivery vector for BMP-2 therapy. These results suggest that treatment with bioartificial integrin-specific PEG hydrogels may be a promising clinical strategy for bone regeneration in large bone defects.

Hyaluronic acid (HA) is a naturally occurring polymer that holds considerable promise for tissue engineering applications. Current cross-linking chemistries often require a coupling agent, catalyst, or photoinitiator, which may be cytotoxic, or involve a multistep synthesis of functionalized-HA, increasing the complexity of the system. With the goal of designing a simpler one-step , aqueous-based cross-linking system, we synthesized HA hydrogels via Diels-Alder “click” chemistry. Furan-modified HA derivates were synthesized and cross-linked via dimaleimide poly(ethylene glycol). By controlling the furan to maleimide molar ratio, both the mechanical and degradation properties of the resulting Diels-Alder cross-linked hydrogels can be tuned. Rheological and degradation studies demonstrate that the Diels-Alder click reaction is a suitable cross-linking method for HA. These HA cross-linked hydrogels were shown to be cytocompatible and may represent a promising material for soft tissue engineering.

Hyaluronic acid (HA) is a naturally occurring polymer that holds considerable promise for tissue engineering applications. Current cross-linking chemistries often require a coupling agent, catalyst, or photoinitiator, which may be cytotoxic, or involve a multistep synthesis of functionalized-HA, increasing the complexity of the system. With the goal of designing a simpler one-step , aqueous-based cross-linking system, we synthesized HA hydrogels via Diels-Alder “click” chemistry. Furan-modified HA derivates were synthesized and cross-linked via dimaleimide poly(ethylene glycol). By controlling the furan to maleimide molar ratio, both the mechanical and degradation properties of the resulting Diels-Alder cross-linked hydrogels can be tuned. Rheological and degradation studies demonstrate that the Diels-Alder click reaction is a suitable cross-linking method for HA. These HA cross-linked hydrogels were shown to be cytocompatible and may represent a promising material for soft tissue engineering.

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.

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.

No description available.

The loss of regenerative capacity of bone, from fetal to adult to aged animals, has been attributed not only to a decline in the function of cells involved in bone formation but also to alterations in the bone microenvironment that occur through development and aging, including extracellular matrix (ECM) composition and growth/trophic factor content. In the development of novel treatments for bone repair, one potential therapeutic goal is the restoration of a more regenerative microenvironment, as found during embryonic development. One approach to creating such a microenvironment is through the use of stem cells. In addition to serving as a differentiated cell source, pluripotent stem cells, such as embryonic stem cells (ESCs), may possess the unique potential to modulate tissue environments via local production of ECM and growth factors. ESC-produced factors may be harnessed and delivered to promote functional tissue regeneration. Such an approach to generate a naturally derived, acelluar therapy has been employed successfully to deliver osteoinductive factors found within adult bone, in the form of demineralized bone matrix (DBM), but the development of treatments derived instead from developing, more regenerative tissues or cells remains attractive. Furthermore, the derivation of regenerative materials from an ESC source also presents the added benefit of eliminating donor to donor variability of adult, cadaveric tissue derived materials, such as DBM. Thus, the objective of this project was to examine the osteoinductive potential harbored within the embryonic microenvironment, in vitro and in vivo. The osteogenic differentiation of mouse ESCs as embryoid bodies (EBs) was evaluated in response to phosphate treatment, in vitro, including osteoinductive growth factor production. The osteoinductivity of EB-derived material (EBM) was then compared to that of adult tissue-derived DBM, in vivo. Phosphate treatment enhanced osteogenic differentiation of EBs. EBM derived from phosphate treated EBs retained bioactive, osteoinductive factors and induced new bone formation, demonstrating that the microenvironment within osteogenic EBs can be harnessed in an acellular material to yield in vivo osteoinductivity. This work not only provides new insights into the dynamic microenvironments of differentiating stem cells but also establishes an approach for the development of an ESC-derived, tissue specific therapy.