Osteoarthritis is a degenerative joint disease characterized by progressive erosion of the articular cartilage. Epidemiological studies have established a relationship between osteoarthritis and menopause suggesting that estrogen may be important in the development of cartilage regeneration therapies. The overall goal of this research project was to advance the field of cartilage tissue regeneration by investigating the role of 17 ß -estradiol (E2), an active estrogen metabolite, on the chondrocyte phenotype. The central hypothesis was that E2 plays an important and sex-specific role in regulating chondrogenesis. Specific Aim-1 focused on establishing and characterizing a primary human articular chondrocyte (HAC) cell source, and then examining the response of the cells in culture to E2. It was demonstrated that the response of HACs to E2 treatment was sex-specific despite both male and females cells expressing estrogen receptors. Female HACs showed changes in proliferation, matrix production, and differentiation while male cells did not. In addition, the female response was regulated through a rapid membrane signaling pathway mediated by protein kinase C. Specific Aim-2 involved establishing an ovariectomized animal model to investigate the effects of E2 on orthopaedic tissue implants. Human demineralized bone matrix (DBM) was implanted intramuscularly into female nude mice and rats. Ovariectomy was shown to reduce the ability of DBM to induce the formation of cartilage and bone tissue. Moreover, the inductive properties of DBM were reestablished with subcutaneous E2 supplementation. Specific Aim-3 entailed developing and characterizing a microencapsulation method for in vitro culture and in vivo delivery of chondrocytes to study the effects of E2 on chondrogenesis. Rat growth plate chondrocytes and HACs were microencapsulated in alginate using an extrusion method in conjunction with high electrostatic potential. Chondrocytes maintained their phenotype in alginate suspension but were unable to form cartilage tissue when implanted into our animal model. Further optimization of the system is required before the role of E2 on chondrogenesis of tissue engineered constructs can be determined. In summary, our results suggest that the successful production of tissue engineered therapies will likely depend on understanding and manipulating the actions of sex hormones in both the in vitro and in vivo environment.

microRNAs (miRNAs), ~21-24 nucleotide-long RNAs that post-transcriptionally regulate gene expression, have rapidly become one of the most extensively studied mechanisms of the past decade. Since their discovery as temporal regulators of post-embryonic development in C. elegans, miRNAs have been functionally implicated in almost every cellular process investigated to date. miRNAs are integral to the complex biological processes of embryonic development and aging. In this research, we sought to determine whether misregulation of miRNAs could be responsible for eliciting adverse effects during these two distinct developmental stages. First, to uncover the potential role of miRNAs in teratogenicity, we investigated whether miRNAs were involved in regulation of retinoic acid (RA) induced vertebrate axis defects. Global miRNA expression profiling revealed that RA exposure suppressed the expression of miR-19 family members during zebrafish somitogenesis. Bioinformatics analyses predict that miR-19 targets cyp26a1, a key RA detoxifying enzyme, and a physiological reporter assay confirmed that cyp26a1 is a bona fide target of miR-19. Transient knockdown of miR-19 phenocopied RA-induced body axis defects. In gain-of-function studies, exogenous miR-19 rescued the axis defects caused by RA exposure. Our findings indicate that the teratogenic effects of RA exposure result, in part, from repression of miR-19 and the subsequent misregulation of cyp26a1. This highlights a previously unidentified role of miR-19 in facilitating vertebrate axis development. Next, to explore whether age-related changes in miRNAs trigger deficits in regeneration capacity, we performed mRNA and small RNA sequencing on regenerating and non-regenerating caudal fin tissue from aged, adult and juvenile zebrafish. An unbiased approach identified cbx7 as the most abundant transcript with significantly increased expression in regenerative-competent adult and juvenile tissue and decreased expression in regenerative-compromised aged tissue. While cbx7 is a known regulator of aging, this is the first report of its role in tissue regeneration. A computational approach was used to discover mRNAs expressed during regeneration, which are potential targets of the significantly expressed miRNAs in regenerating tissue. miR-21 was one of the most abundant and significantly increased miRNAs in regenerating tissue and exhibited an aberrant age-dependent expression profile. Bioinformatics predicts miR-21 to target the 3' UTR of cbx7 and a reporter assay confirmed that miR-21 targets cbx7 in vivo. Transient knockdown of miR-21 inhibited tissue regeneration, suggesting a role for miRNA mediated regulation of cbx7 during regeneration. These findings reveal a novel, age-dependent regenerative function of cbx7 and emphasize the importance of miR-21 as a master regulator of vertebrate regenerative responses. This research, when combined, underscores the negative consequences misregulation of miRNAs has on embryonic development and aging. / Graduation date: 2013 / Access restricted to the OSU Community at author's request from Dec. 5, 2012 - Dec. 5, 2013

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.

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.

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.

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.

In response to high and rising amputation rates, significant advances have been made in the field of prosthetic limb design. Unfortunately, there exists a lag in the neural interfacing technology required to provide an adequate link between the nervous system and this emerging generation of advanced prosthetic devices. Novel approaches to peripheral nerve interfacing are required to establish the stable, high channel count connections necessary to provide natural, thought driven control of an external prosthesis. Here, a tissue engineering-based approach has been used to create a device capable of interfacing with a regenerated portion of amputated nerve. As part of this work, a nerve guidance channel design, in which small amounts of interior scaffolding material could be precisely positioned, was evaluated. Guidance channels containing a single thin-film sheet of aligned scaffolding were shown to support robust functional nerve regeneration across extended injury gaps by minimally supplementing natural repair mechanisms. Significantly, these "thin-film enhanced nerve guidance channels" also provided the capability to guide the course of axons regenerating from a cut nerve. This capability to control axonal growth was next leveraged to create "regenerative scaffold electrodes (RSEs)" able to interface with axons regenerating from an amputated nerve. In the RSE design, low-profile arrays of interfacing electrodes were embedded within layers of aligned scaffolding material, such that regenerating axons were topographically guided by the scaffolding through the device and directly across the embedded electrodes. Chronically implanted RSEs were successfully used to record evoked neural activity from amputated nerves in an animal model. These results demonstrate that the use of topographic cues within a nerve guidance channel might offer the potential to influence the course of nerve regeneration to the advantage of a peripheral nerve interface suitable for limb amputees.

A queimadura de 3º grau é um dos maiores traumas a que um ser humano pode ser submetido e em geral são as lesões mais frequentes na população mundial, tratando-se de um importante problema de saúde pública. Pensando neste problema, observou-se que a utilização da membrana amniótica (MA) pode ser o melhor tratamento que esses pacientes possam receber. Ela atua em benefício da epitelização, facilita a migração e a adesão das células epiteliais basais, a matriz estromal possui inibidores de proteases que previne a apoptose e restaura o fenótipo epitelial, antibacteriana do córion e âmnion, além de proteger a ferida e atuar na redução da dor. A descelularização, trata-se da retirada de todas as células e núcleo da MA utilizada, como forma de se evitar qualquer tipo de histoincompatibilidade. O presente trabalho teve como objetivo avaliar a eficácia do uso da membrana amniótica descelularizada (MAD) no tratamento de queimaduras de 3o grau em humanos, testada em ratos da linhagem Wistar. Trata-se de uma pesquisa descritiva, a qual investiga fundamentalmente a especificação e a disposição de dados acerca dos resultados obtidos a partir de um processo regenerativo de queimaduras utilizando-se a MAD. A amostra foi constituída de constituída por 20 ratos machos adultos da linhagem Wistar, dividida em 2 grupos (n=10): grupo controle (GC)–sem MA e grupo transplantado–com MA (TMAD). Os dois grupos foram submetidos a um processo padronizado de queimadura térmica, sendo retiradas duas amostras de tecido para análise, no 14º dia e no 30º dia. As amostras foram analisadas com o emprego de técnicas anatomopatológicas onde foram montadas em lâminas fragmentos de MA corados com solução Hematoxilina Eosina (HE) e, para análise histomorfométrica,as lâminas foram coradas com picro sirius red sob luz polarizada para verificação da descelularização e quantificação de colágeno do tipo I, II e III. As imagens foram quantificadas utilizando o programa Image Pro Plus®, versão 5.0 para Windows®. Os dados estatísticos foram analisados utilizando o programa computacional SSPS v.21.0. Observou-se um aumento significativo da quantificação de colágenos do tipo III no 14º dia no grupo TMAD em relação ao GC, do colágeno do tipo II no 14º e 30º dia comparado ao GC e do colágeno tipo I no 14º e 30º dia, mostrando que a utilização da MA foi eficaz no grupo TMAD. Concluiu-se que a MAD, aplicada topicamente, demonstrou eficácia no processo cicatricial em queimaduras de 3º grau. Esperava-se que a mesma promovesse a aceleração de cicatrização dos ferimentos, mas, o que se observou ao término do trabalho é que a MAD não só promoveu a cicatrização como foi mais eficiente no processo de regeneração dos tecidos lesados. / 3rd degree of burns are one of the greatest injury that a human being can be submitted and generally are the most frequent injuries in the world population, in the case of a major public health problem. Thinking this problem, it has been observed that the use of amniotic membrane (AM) may be the best treatment these patients can receive. It acts on behalf of epithelialization, facilitates migration and adhesion of basal epithelial cells, stromal matrix has protease inhibitors which prevent apoptosis and restores epithelial, antibacterial phenotype of the chorion and amnion, and protect the wound and act on reducing pain. The decellularization, it is the withdrawal of all cells and core AM used as a way to avoid any kind of histoincompatibility. This study aimed to evaluate the efficacy of the decellularized amniotic membrane (DAM) in the treatment of third degree burns in humans, tested in Wistar rats. It is a descriptive research which investigates mainly the specification and the arrangement of data on the results obtained from a regenerative process burns using DAM. The sample consisted of consisted of 20 Wistar adult male rats, divided into 2 groups (n = 10): control group (CG) - without MA, and group transplanted - with AM (TDAM). The two groups were submitted to a standard process of thermal burn, two samples being removed tissue for analysis on day 14 and day 30. Samples were analyzed with the use of pathological techniques which were mounted on slides DNA fragments stained with solution hematoxylin eosin (HE) for histomorphometric analysis, the slides were stained with picrosirius red under polarized light for verification of decellularization and quantifying collagen type I, II and III. Images were quantified using Image Pro Plus ®, version 5.0 for Windows. Statistical data were analyzed using the computer program SPSS v.21.0. There was a significant increase in type III collagen quantification on the 14th day in TDAM group compared to CG, type II collagen in the 14th and 30th day compared to the CG and type I collagen in the 14th and 30th day, showing that the use of MA were effective in TDAM group. It was concluded that the DAM, applied topically, has shown efficacy in the healing process in 3rd degree burns. It was expected that it would promote accelerated wound healing but which was observed at the end of work that MAD is not only promoted as the healing was more efficient the regeneration process of the damaged tissues.

Indiana University-Purdue University Indianapolis (IUPUI) / Hydrogels prepared from photopolymerization have been used for a variety of tissue engineering and controlled release applications. Polymeric biomaterials with high cytocompatibility, versatile degradation behaviors, and diverse material properties are particularly useful in studying cell fate processes. In recent years, step-growth thiol-ene photochemistry has been utilized to form cytocompatible hydrogels for tissue engineering applications. This radical-mediated gelation scheme utilizes norbornene functionalized multi-arm poly(ethylene glycol) (PEGNB) as the macromer and di-thiol containing molecules as the crosslinkers to form chemically crosslinked hydrogels. While the gelation mechanism was well-described in the literature, the network properties and degradation behaviors of these hydrogels have not been fully characterized. In addition, existing thiol-ene photopolymerizations often used type I photoinitiators in conjunction with an ultraviolet (UV) light source to initiate gelation. The use of cleavage type initiators and UV light often raises biosafety concerns. The first objective of this thesis was to understand the gelation and degradation properties of thiol-ene hydrogels. In this regard, two types of step-growth hydrogels were compared, namely thiol-ene hydrogels and Michael-type addition hydrogels. Between these two step-growth gel systems, it was found that thiol-ene click reactions formed hydrogels with higher crosslinking efficiency. However, thiol-ene hydrogels still contained significant network non-ideality, demonstrated by a high dependency of hydrogel swelling on macromer contents. In addition, the presence of ester bonds within the PEGNB macromer rendered thiol-ene hydrogels hydrolytically degradable. Through validating model predictions with experimental results, it was found that the hydrolytic degradation of thiol-ene hydrogels was not only governed by ester bond hydrolysis, but also affected by the degree of network crosslinking. In an attempt to manipulate network crosslinking and degradation rate of thiol-ene hydrogels, different macromer contents and peptide crosslinkers with different amino acid sequences were used. A chymotrypsin-sensitive peptide was also used as part of the hydrogel crosslinkers to render thiol-ene hydrogels enzymatically degradable. The second objective of this thesis was to develop a visible light-mediated thiol-ene hydrogelation scheme using a type II photoinitiator, eosin-Y, as the only photoinitiator. This approach eliminates the incorporation of potentially cytotoxic co-initiator and co-monomer that are typically used with a type II initiator. In addition to investigating the gelation kinetics and properties of thiol-ene hydrogels formed by this new gelation scheme, it was found that the visible light-mediated thiol-ene hydrogels were highly cytocompatible for human mesenchymal stem cells (hMSCs) and pancreatic MIN6 beta-cells. It was also found that eosin-Y could be repeatedly excited for preparing step-growth hydrogels with multilayer structures. This new gelation chemistry may have great utilities in controlled release of multiple sensitive growth factors and encapsulation of multiple cell types for tissue regeneration.

Indiana University-Purdue University Indianapolis (IUPUI) / Bone growth can be impaired due to disease, such as osteoporosis. Currently, intermittent parathyroid hormone (PTH) treatment is the only approved therapy in the United States for anabolic bone growth in osteoporosis patients. The anabolic effects of PTH treatment are due, at least in part, to modulation of the Wnt/β-catenin pathway. Activation of the Wnt/ β-catenin pathway using a small molecule inhibitor of GSK3β was previously shown to increase markers of bone formation in vitro. Our study utilized a zebrafish model system to study Wnt activated fin regeneration and bone growth. Wnt signaling is the first genetically identified step in fin regeneration, and bony rays are the main structure in zebrafish fins. Thus, zebrafish fin regeneration may be a useful model to study Wnt signaling mediated bone growth. Fin regeneration experiments were conducted using various concentrations of a GSK3β inhibitor compound, LSN 2105786, for different treatment periods and regenerative outgrowth was measured at 4 and 7 days post amputation. Experiments revealed continuous low concentration (4-5 nM) treatment to be most effective at increasing regeneration. Higher concentrations inhibited fin growth, perhaps by excessive stimulation of differentiation programs. In situ hybridization experiments were performed to examine effects of GSK3β inhibitor on Wnt responsive gene expression. Experiments showed temporal and spatial changes on individual gene markers following GSK3β inhibitor treatment. Additionally, confocal microscopy and immunofluorescence labeling data indicated that the Wnt signaling intracellular signal transducer, β-catenin, accumulates throughout GSK3β inhibitor treated tissues. Finally, experiments revealed increased cell proliferation in fin regenerates following LSN 2105786 treatment. Together, these data indicate that bone growth in zebrafish fin regeneration is improved by activating Wnt signaling. Zebrafish Wnt signaling experiments provide a good model to study bone growth and bone repair mechanisms, and may provide an efficient drug discovery platform.