Jennifer E. Phillips

CTCF: Master Weaver of the Genome

CTCF is a highly conserved zinc finger protein implicated in diverse regulatory functions, including transcriptional activation/repression, insulation, imprinting, and X chromosome inactivation. Here we re-evaluate data supporting these roles in the context of mechanistic insights provided by recent genome-wide studies and highlight evidence for CTCF-mediated intra- and interchromosomal contacts at several developmentally regulated genomic loci. These analyses support a primary role for CTCF in the global organization of chromatin architecture and suggest that CTCF may be a heritable component of an epigenetic system regulating the interplay between DNA methylation, higher-order chromatin structure, and lineage-specific gene expression.

Engineering graded tissue interfaces

Interfacial zones between tissues provide specialized, transitional junctions central to normal tissue function. Regenerative medicine strategies focused on multiple cell types and/or bi/tri-layered scaffolds do not provide continuously graded interfaces, severely limiting the integration and biological performance of engineered tissue substitutes. Inspired by the bone-soft tissue interface, we describe a biomaterial-mediated gene transfer strategy for spatially regulated genetic modification and differentiation of primary dermal fibroblasts within tissue-engineered constructs. We demonstrate that zonal organization of osteoblastic and fibroblastic cellular phenotypes can be engineered by a simple, one-step seeding of fibroblasts onto scaffolds containing a spatial distribution of retrovirus encoding the osteogenic transcription factor Runx2/Cbfa1. Gradients of immobilized retrovirus, achieved via deposition of controlled poly(l-lysine) densities, resulted in spatial patterns of transcription factor expression, osteoblastic differentiation, and mineralized matrix deposition. Notably, this graded distribution of mineral deposition and mechanical properties was maintained when implanted in vivo in an ectopic site. Development of this facile and robust strategy is significant toward the regeneration of continuous interfacial zones that mimic the cellular and microstructural characteristics of native tissue.

Human mesenchymal stem cell differentiation on self-assembled monolayers presenting different surface chemistries

Human mesenchymal stem cells (hMSCs) have tremendous potential as a cell source for regenerative medicine due to their capacity for differentiation into a wide range of connective tissue cell types. Although significant progress has been made in the identification of defined growth factor conditions to induce lineage commitment, the effect of underlying biomaterial properties on functional differentiation is far less understood. Here we conduct a systematic assessment of the role for surface chemistry on cell growth, morphology, gene expression and function during hMSC commitment along osteogenic, chondrogenic and adipogenic lineages. Using self-assembled monolayers of omega-functionalized alkanethiols on gold as model substrates, we demonstrate that biomaterial surface chemistry differentially modulates hMSC differentiation in a lineage-dependent manner. These results highlight the importance of initial biomaterial surface chemistry on long-term functional differentiation of adult stem cells, and suggest that surface properties are a critical parameter that must be considered in the design of biomaterials for stem cell-based regenerative medicine strategies.

Glucocorticoid-induced osteogenesis is negatively regulated by Runx2/Cbfa1 serine phosphorylation

Glucocorticoid hormones have complex stimulatory and inhibitory effects on skeletal metabolism. Endogenous glucocorticoid signaling is required for normal bone formation in vivo, and synthetic glucocorticoids, such as dexamethasone, promote osteoblastic differentiation in several in vitro model systems. The mechanism by which these hormones induce osteogenesis remains poorly understood. We demonstrate here that the coordinate action of dexamethasone and the osteogenic transcription factor Runx2/Cbfa1 synergistically induces osteocalcin and bone sialoprotein gene expression, alkaline phosphatase activity, and biological mineral deposition in primary dermal fibroblasts. Dexamethasone decreased Runx2 phosphoserine levels, particularly on Ser125, in parallel with the upregulation of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) through a glucocorticoid-receptor-mediated mechanism. Inhibition of MKP-1 abrogated the dexamethasone-induced decrease in Runx2 serine phosphorylation, suggesting that glucocorticoids modulate Runx2 phosphorylation via MKP-1. Mutation of Ser125 to glutamic acid, mimicking constitutive phosphorylation, inhibited Runx2-mediated osteoblastic differentiation, which was not rescued by dexamethasone treatment. Conversely, mutation of Ser125 to glycine, mimicking constitutive dephosphorylation, markedly increased osteoblastic differentiation, which was enhanced by, but did not require, additional dexamethasone supplementation. Collectively, these results demonstrate that dexamethasone induces osteogenesis, at least in part, by modulating the phosphorylation state of a negative-regulatory serine residue (Ser125) on Runx2. This work identifies a novel mechanism for glucocorticoid-induced osteogenic differentiation and provides insights into the role of Runx2 phosphorylation during skeletal development.

Analyzing bone, blood vessels, and biomaterials with microcomputed tomography

Quantitative tools such as micro-CT are needed for tissue engineering to evolve beyond a qualitative, observational field and accelerate the clinical realization of regenerative technologies. As faster, higher-resolution micro-CT systems become available for both in vitro and in vivo studies and the development of improved contrast agents allows micro-CT imaging to be extended to nonmineralized tissues, additional novel applications related to tissue engineering are sure to emerge.

Retroviral-Mediated Gene Therapy for the Differentiation of Primary Cells into a Mineralizing Osteoblastic Phenotype

Bone tissue engineering has emerged as a promising strategy for the repair of critical-sized skeletal fractures. However, the clinical application of this approach has been limited by the availability of a robust mineralizing cell source. Non-osteogenic cells, such as skin fibroblasts, are an attractive cell-source alternative because they are easy to harvest from autologous donor skin biopsies and display a high capacity for in vitro expansion. We have recently demonstrated that retroviral gene delivery of the osteoblastic transcription factor Runx2/Cbfa1 promotes osteogenic differentiation in primary dermal fibroblasts cultured in monolayer. Notably, sustained expression of Runx2 was not sufficient to promote functional osteogenesis in these cells, and co-treatment with the steroid hormone dexamethasone was required to induce deposition of biologically-equivalent matrix mineralization. On the basis of these results, we then investigated the osteogenic capacity of these genetically engineered fibroblasts when seeded on polymeric scaffolds in vitro and in vivo. These experiments demonstrated that Runx2-expressing fibroblasts seeded on collagen scaffolds produce significant levels of matrix mineralization after 28 days in vivo implantation in a subcutaneous, heterotopic site. Overall, these results offer evidence that transcription factor-based gene therapy may be a powerful strategy for the conversion of a non-osteogenic cellular phenotype into a mineralizing cell source for bone repair applications. This concept may also be applied to control functional differentiation in a broad range of cell types and tissue engineering applications. The chapter below outlines detailed methods for the isolation and ex vivo genetic modification of primary dermal fibroblasts using retroviral-mediated delivery of the Runx2 transgene in both monolayer culture and three-dimensional scaffolds.

Characterization of a small animal growth plate injury model using microcomputed tomography.

Injuries to the growth plate remain a significant clinical challenge. The need to better understand mechanisms of growth disruption following transphyseal injuries and evaluate new therapeutic approaches to growth restoration motivates development of a well characterized model of growth plate injury. The goals of this study were to develop a growth plate defect model in the rat and to use microcomputed tomography (micro-CT) imaging to detect and quantify associated changes in growth plate morphology and mineralization over time following injury and in response to treatment. Three-dimensional images of the growth plate were created from micro-CT scans and used to quantify the volume of mineralized tissue within the defect site. Growth plate thickness and volume as well as the degree of growth plate fusion were also measured from the reconstructed 3D images. Growth deficiency was then quantified as a function of time post-injury from whole limb micro-CT scans. Finally, this model was used to determine the ability of an injectable in situ gelling hydrogel to prevent formation of a bony bridge within the defect and the subsequent effect on limb length deficiency and changes to growth plate morphology. Growth plate injury resulted in significant shortening of the defect limb by day 28 and significant thinning and fusion of the surrounding growth plate up to day 112. Limb length reduction was correlated with changes in the growth plate volume and average thickness at day 56. Injection of an in situ gelling agarose into the defect resulted in a reduction of limb length discrepancy as well as a thicker growth plate on average compared to empty defect controls. These results establish a novel method of characterizing changes in whole bone and growth plate morphology due to a growth plate injury and indicate that treatment with agarose hydrogel reduces limb length discrepancy but is not sufficient to regenerate growth plate tissue or fully restore growth function.

Intraabdominal adhesion formation after preperitoneal dissection in the murine model

AbstractBackground: The laparoscopic approach to hernia repair has been advocated by many as a potentially superior method of herniorraphy. Several techniques have been described, each with its own proposed advantages. These techniques involve different anatomic approaches, the most recent of which is the totally extraperitoneal approach (TEPA). One presumed advantage of the extraperitoneal approach is the avoidance of adhesion formation because the peritoneum is not entered and mesh is not placed in direct contact with intra-abdominal structures. We hypothesize, however, that when the peritoneum is dissected from the abdominal wall, it is partially devascularized, leading to scar formation and potential adhesion formation. This would suggest that the TEPA method of herniorraphy may not completely avoid the risks of intra-abdominal adhesion formation. Methods: After appropriate approval was obtained, 88 male Sprague-Dawley rats were divided into two equal groups. One group underwent laparotomy followed by careful blunt dissection of the peritoneum from the left abdominal wall. The control group underwent laparotomy without manipulation of the peritoneum. All animals were re-explored 14 days later, and the abdominal cavity was examined for adhesions. The type and location of any adhesion was recorded. Results: Adhesion formation occurred in 10 of 44 (23%) subjects in the peritoneal dissection group, compared with 3 of 44 (7%) in the nondissection group (p < 0.05). Conclusions: Dissection of the peritoneum from the overlying abdominal wall in the murine model leads to intra-abdominal adhesion formation. This suggests that peritoneal dissection in the TEPA method of herniorraphy may lead to intra-abdominal adhesion formation.

Addressing cell-sourcing limitations with gene therapy

Genetic engineering with Run/spl times/2/Cbfa 1 for an alternative to biological grafts. Forced Runx2 expression via retroviral gene delivery promotes osteoblastic differentiation and mineralization in bone marrow stromal cells and stimulates conversion of myoblasts and dermal fibroblasts into a mineralizing osteoblastic phenotype. This genetic engineering strategy provides a promising approach to overcome cell-sourcing limitations in bone tissue engineering.