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.

Large-scale expansion of lineage-committed stem cells can provide an excellent ex vivo model for studying complex molecular pathways governing cell fate choices. Also, such cells could be useful for implementing cell therapeutic approaches for treatment of specific disorders involving extensive cellular damage within that lineage. Using growth factors, pluri- and multipotent stem cells have been successfully isolated and cultured from pre- and peri-implantation stage embryos, including trophectoderm, primitive ectoderm, epiblast and primitive endoderm. However, ex vivo expansion of lineage restricted cells from later embryonic lineages and adult tissues have been a challenge. N-myc is a well-characterized member of myc gene family that is known to be essential for the proliferation of numerous progenitor cell types during normal embryonic development of diverse organs including lungs, liver, heart, kidneys and brain. Considering this important role of N-myc, we hypothesized that its regulated activation in these progenitors might allow their expansion in culture. To test this hypothesis, we generated a novel doxycycline-inducible transgenic mouse line that expresses N-myc uniformly across all tissues. Using cortical precursors derived from mid-gestation embryos of these mice, we show that upon doxycycline induced N-myc expression, we can achieve at least a million-fold expansion of multipotent neural precursors within a short span of time in culture. When doxycycline is withdrawn, N-myc expression is turned off and the cells differentiate into neurons and glia. An extensive characterization of the expanded cells revealed that the cells retained their differentiation potential, genomic stability and commitment specific to their origin. The tetracycline-inducible N-myc expressing mouse line might also serve as a source for establishing other than neural lineage committed progenitor cell lines where N-myc has a known role in regulating cell proliferation and differentiation decisions.

Large-scale expansion of lineage-committed stem cells can provide an excellent ex vivo model for studying complex molecular pathways governing cell fate choices. Also, such cells could be useful for implementing cell therapeutic approaches for treatment of specific disorders involving extensive cellular damage within that lineage. Using growth factors, pluri- and multipotent stem cells have been successfully isolated and cultured from pre- and peri-implantation stage embryos, including trophectoderm, primitive ectoderm, epiblast and primitive endoderm. However, ex vivo expansion of lineage restricted cells from later embryonic lineages and adult tissues have been a challenge. N-myc is a well-characterized member of myc gene family that is known to be essential for the proliferation of numerous progenitor cell types during normal embryonic development of diverse organs including lungs, liver, heart, kidneys and brain. Considering this important role of N-myc, we hypothesized that its regulated activation in these progenitors might allow their expansion in culture. To test this hypothesis, we generated a novel doxycycline-inducible transgenic mouse line that expresses N-myc uniformly across all tissues. Using cortical precursors derived from mid-gestation embryos of these mice, we show that upon doxycycline induced N-myc expression, we can achieve at least a million-fold expansion of multipotent neural precursors within a short span of time in culture. When doxycycline is withdrawn, N-myc expression is turned off and the cells differentiate into neurons and glia. An extensive characterization of the expanded cells revealed that the cells retained their differentiation potential, genomic stability and commitment specific to their origin. The tetracycline-inducible N-myc expressing mouse line might also serve as a source for establishing other than neural lineage committed progenitor cell lines where N-myc has a known role in regulating cell proliferation and differentiation decisions.

Vascularization of tissue-engineered substitutes is imperative for successful implantation into sites of injury. Strategies to promote vascularization within tissue-engineered constructs have focused on incorporating endothelial or endothelial progenitor cells within the construct. However, since endothelial and endothelial progenitor cells are adult cell types and limited in number, acquiring quantities needed for regenerative medicine applications is not feasible. Pluriopotent stem cells have been explored as a cell source for tissue-engineered substitutes because of their inherent ability to differentiate into all somatic cell types, including endothelial cells (ECs). Current EC differentiation strategies require laborious and extensive culture periods, utilize large quantities of expensive growth factors and extracellular matrix, and generally yield heterogenous populations for which only a small percentage of the differentiated cells are ECs. In order to recapitulate in vivo embryonic stem cell (ESC) differentiation, 3D stem cell aggregates or embryoid bodies (EBs) have been employed in vitro. In the developing embryo, fluid shear stress, VEGF, and oxygen are instructive cues for endothelial differentiation and vasculogenesis. Thus, the objective of this work was to study the effects of fluid shear stress pre-conditioning of ESCs on EB endothelial differentiation and vasculogensis. The overall hypothesis is that exposing ESCs to fluid shear stress prior to EB differentiation will promote EB endothelial differentiation and vasculogenesis. Pre-conditioning ESCs with fluid shear stress modulated EB differentiation as well as endothelial cell-like cellular organization and EB morphogenesis. To further promote endothelial differentiation, ESCs pre-conditioned with shear were treated with VEGF. Exposing EBs formed from ESCs pre-conditioned with shear to low oxygen resulted in increased production of VEGF and formation of endothelial networks. The results of this work demonstrate the role that physical forces play in modulating stem cell fate and morphogenesis.

The myocardial infarction (MI) is one of the leading causes of death in the world today. Accumulated atherosclerotic plaque occluding cardiac blood vessels results in a lack of oxygen supply to parts of the heart, and consequentially the death cardiomyocytes. The damaged area is replaced by scar tissue because of the heart’s insufficient regenerative capability, and the contraction property of the post-MI heart is therefore compromised. The recent findings of an endogenous cardiac progenitor cell (CPC) population gives hope for the establishment of new methods for medical treatments of the post-MI heart. Compared to other stem/progenitor cell sources, the CPCs are committed to a cardiac fate which places them in the forefront of interesting cell sources for regenerative treatments. In this thesis, the distribution of stem cell antigen 1 (Sca-1) positive CPCs in the healthy mouse myocardium, as well as the healthy and post-MI rat left ventricle was determined and compared to the total amount of nuclei. An immunohistochemistry protocol for the detection of Sca-1+ cells was established, and the number of Sca-1+ cells and the total number of nuclei in the different mouse and rat tissue samples were counted using laser scanning cytometry (LSC). The results could conclude a significantly higher distribution of Sca-1+ cells in the mouse atrium compared to the mouse ventricle, and a significantly higher distribution of Sca-1+ cells in the 8 days post-MI rat left ventricle compared to the healthy rat left ventricle. Furthermore, a heterogeneous distribution within the 8 days post-MI rat left ventricle was observed.

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.

Embryonic stem cells (ESCs) hold great promise for treatment of degenerative disorders such as Parkinson's and Alzheimer's disease, diabetes, and cardiovascular disease. The ability of ESCs to differentiate to all somatic cell types suggests that they may serve as a robust cell source for production of differentiated cells for regenerative medicine and other cell-based therapeutics. In order for ESCs to be used effectively in clinical settings, efficient and reproducible differentiation to targeted cell types must be demonstrated. The overall objective of this project was to engineer microenvironmental control over differentiating ESCs through the formation of embryoid bodies (EBs) uniform in size and shape, and through the incorporation of morphogen-containing polymer microspheres within the interior of EBs. The central hypothesis was that morphogen delivery through incorporated polymer microspheres within a uniform population of EBs will induce controlled and uniform differentiation of ESCs. Rotary suspension culture was developed in order to efficiently produce uniform EBs in high yield. Compared to static suspension culture, rotary suspension significantly improved the production of differentiating cells and EBs over the course of 7 days, while simultaneously improving the homogeneity of EB size and shape compared to both hanging drop and static EBs. The diffusive transport properties of EBs formed via rotary suspension were investigated using a fluorescent, cell permeable dye to model the movement of small morphogenic molecules within EBs. Confocal microscopy, cryosections and EB dissociation all demonstrated that the dye was not able to fully penetrate EB, and that the larger EBs at later time points (7 days) retarded dye movement to a greater extent than earlier EBs (days 2 and 4). Polymer microspheres capable of encapsulating morphogenic factors were incorporated into EBs in order to overcome the diffusional limitations of traditional soluble delivery. The size of microspheres, microsphere coating, microsphere to cell ratio, and rotary mixing speed were all observed to influence incorporation within EBs. The use of microsphere-mediated delivery within EBs to direct cell differentiation was examined. Microsphere-mediated delivery of retinoic acid (RA) induced formation of uniquely cystic spheroids with a visceral endoderm layer enveloping a pseudo-stratified columnar epithelium, and with spatial localization of transcriptional profiles similar to the early primitive streak stage of mouse development. Continued differentiation of RA MS EBs in defined media conditions was assessed. Gene expression demonstrated that regular serum enhanced endoderm induction, serum-free media supported ectoderm differentiation, while mesoderm was most prominent in untreated EBs in full serum. In summary, this work has realized a unique approach for stem cell differentiation through modification of the internal microenvironment of ESC spheroids. This novel inside-out method toward engineering EBs demonstrated that the mode of morphogen delivery significantly affected the course of differentiation. These studies provide the basis for ongoing work, which will utilize the choice of microsphere material, coating, and morphogen in order to uniquely study mechanisms of ESC differentiation and achieve unparalleled engineering of the EB microenvironment.

It has been shown that stem cells exist within adult adipose tissue. These stem cells are named adipose-derived mesenchymal stem cells (ASCs), are derived from the mesoderm, and can differentiate into a number of cells including osteoblasts, chondrocytes, and adipocytes. However, before these cells can be used clinically it is important that we understand how factors like age, sex, and ethnicity affect ASC number and potential. Additionally, since men and women vary in their distribution of adipose tissue, it will be important to see if the ideal source of ASCs is different for each sex. The goal of this study was to assess how age and sex affects ASCs. We used flow cytometry to investigate how age and sex affected the number of ASCs in adipose tissue. Additionally, we plated these cells in culture and treated them with an osteogenic media (OM) with the intention of pushing them towards osteoblast differentiation. The purpose of this was to see if age or sex affected the potential of the ASCs to undergo osteogenesis in culture. For this study we used real-time PCR and biochemical assays to look at markers of early and late osteogenic differentiation. Finally, we used immunohistochemistry to demonstrate where in adipose tissue the CD73 and CD271 positive cell population exists. It is our hope that this work will shed light on how age and sex affect ASCs so that clinicians can optimize their ASC harvest depending on the patient's physiology.

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.

Thesis (Ph.D)--Biomedical Engineering, Georgia Institute of Technology, 2008. / Committee Chair: Joseph Le Doux; Committee Member: Anthanassios Sambanis; Committee Member: David Archer; Committee Member: Michelle LaPlaca; Committee Member: Steve Stice; Committee Member: Todd McDevitt. Part of the SMARTech Electronic Thesis and Dissertation Collection.