BMP2 commitment to the osteogenic lineage involves activation of Runx2 by DLX3 and a homeodomain transcriptional network

Several homeodomain (HD) proteins are critical for skeletal patterning and respond directly to BMP2 as an early step in bone formation. RUNX2, the earliest transcription factor proven essential for commitment to osteoblastogenesis, is also expressed in response to BMP2. However, there is a gap in our knowledge of the regulatory cascade from BMP2 signaling to the onset of osteogenesis. Here we show that BMP2 induces DLX3, a homeodomain protein that activates Runx2 gene transcription. Small interfering RNA knockdown studies in osteoblasts validate that DLX3 is a potent regulator of Runx2. Furthermore in Runx2 null cells, DLX3 forced expression suffices to induce transcription of Runx2, osteocalcin, and alkaline phosphatase genes, thus defining DLX3 as an osteogenic regulator independent of RUNX2. Our studies further show regulation of the Runx2 gene by several homeodomain proteins: MSX2 and CDP/cut repress whereas DLX3 and DLX5 activate endogenous Runx2 expression and promoter activity in non-osseous cells and osteoblasts. These HD proteins exhibit distinct temporal expression profiles during osteoblast differentiation as well as selective association with Runx2 chromatin that is related to Runx2 transcriptional activity and recruitment of RNA polymerase II. Runx2 promoter mutagenesis shows that multiple HD elements control expression of Runx2 in relation to the stages of osteoblast maturation. Our studies establish mechanisms for commitment to the osteogenic lineage directly through BMP2 induction of HD proteins DLX3 and DLX5 that activate Runx2, thus delineating a transcriptional regulatory pathway mediating osteoblast differentiation. We propose that the three homeodomain proteins MSX2, DLX3, and DLX5 provide a key series of molecular switches that regulate expression of Runx2 throughout bone formation. <br/

Chondrogenic Differentiation of Human Mesenchymal Stem Cells in Three-Dimensional Alginate Gels

We characterized the temporal changes in chondrogenic genes and developed a staging scheme for in vitro chondrogenic differentiation of human mesenchymal stem cells (hMSCs) in three-dimensional (3D) alginate gels. A time-dependent accumulation of glycosaminoglycans, aggrecan, and type II collagen was observed in chondrogenic but not in basal constructs over 24 days. qRT-PCR demonstrated a largely characteristic temporal pattern of chondrogenic markers and provided a basis for staging the cellular phenotype into four stages. Stage I (days 0–6) was defined by collagen types I and VI, Sox 4, and BMP-2 showing peak expression levels. In stage II (days 6–12), gene expression for cartilage oligomeric matrix protein, HAPLN1, collagen type XI, and Sox 9 reached peak levels, while gene expression of matrilin 3, Ihh, Homeobox 7, chondroadherin, and WNT 11 peaked at stage III (days 12–18). Finally, cells in stage IV (days 18–24) attained peak levels of aggrecan; collagen IX, II, and X; osteocalcin; fibromodulin; PTHrP; and alkaline phosphatase. Gene profiles at stages III and IV were analogous to those in juvenile articular and adult nucleus pulposus chondrocytes. Gene ontology analyses also demonstrated a specific expression pattern of several putative novel marker genes. These data provide comprehensive insights on chondrogenesis of hMSCs in 3D gels. The derivation of this staging scheme may aid in defining maximally responsive time points for mechanobiological modulation of constructs to produce optimally engineered tissues.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63385/1/tea.2007.0272.pd

Understanding the Chondrogenic Potential of Articular Chondrocytes

Articular cartilage is a smooth, visco-elastic, aneural, avascular tissue made of water, an exquisitely organized framework of proteoglycans, glycosaminoglycans, and collagen fibrils and articular chondrocytes. It’s beautiful organization and composition provide it with the flexibility and strength to cover, protect and lubricate the ends of long bones in a diarthrodial joint. Cartilage homeostasis relies on articular chondrocytes to translate the mechanical forces of daily activity into efficient remodeling of the extracellular matrix. Age, joint injury, or other insulting factors can progressively incapacitate articular chondrocytes, resulting in cartilage lesions that devolve to degenerative joint disease. Therefore, the central idea explored in this dissertation is the changing chondrogenic potential of articular chondrocytes. In the first study, we asked if chondrogenic potential affects how primary articular chondrocytes respond to dynamic Ca2+ signaling, the primary signaling mediator of mechanotransduction during extracellular matrix remodeling. In the second study, we explored how age and culture conditions that alter chondrogenic potential influence the transcriptional profile of primary articular chondrocytes using in-depth RNA-sequencing technology. These studies highlight that the chondrogenic potential of articular chondrocytes, which is affected by age and the gradual changes in matrix composition, can be understood through dynamic signaling and transcriptional networks and enhanced through tissue engineering principles to improve upon the long-term efficacies of cartilage resurfacing procedures

The role of changes in extracellular matrix of cartilage in the presence of inflammation on the pathology of osteoarthritis.

Osteoarthritis (OA) is a degenerative disease that affects various tissues surrounding joints such as articular cartilage, subchondral bone, synovial membrane, and ligaments. No therapy is currently available to completely prevent the initiation or progression of the disease partly due to poor understanding of the mechanisms of the disease pathology. Cartilage is the main tissue afflicted by OA, and chondrocytes, the sole cellular component in the tissue, actively participate in the degeneration process. Multiple factors affect the development and progression of OA including inflammation that is sustained during the progression of the disease and alteration in biomechanical conditions due to wear and tear or trauma in cartilage. During the progression of OA, extracellular matrix (ECM) of cartilage is actively remodeled by chondrocytes under inflammatory conditions. This alteration of ECM, in turn, changes the biomechanical environment of chondrocytes, which further drives the progression of the disease in the presence of inflammation. The changes in ECM composition and structure also prevent participation of mesenchymal stem cells in the repair process by inhibiting their chondrogenic differentiation. This review focuses on how inflammation-induced ECM remodeling disturbs cellular activities to prevent self-regeneration of cartilage in the pathology of OA

Single Cell Molecular Heterogeneity In Musculoskeletal Differentiation

Mesenchymal stem cells (MSCs) display substantial cell-to-cell variation that manifests across many aspects of cell phenotype and complicates the use of MSCs in regenerative applications. However, most conventional assays measure MSC properties in bulk and, as a consequence, mask this cell-to-cell variation. To better understand MSC heterogeneity and its underlying mechanisms, we quantitatively assessed MSC phenotype within the context of chondrogenesis, amongst clonal populations and single cells. Clonal MSCs differed in their contractility, ability to transmit extracellular strain the nucleus, capacity to form cartilage-like matrix, and transcriptomic signature. RNA FISH measurements of single cell gene expression found that both primary chondrocytes and chondrogenically-induced MSCs showed substantial mRNA expression heterogeneity. Surprisingly, variation in differentiation marker transcript levels only weakly associated with cartilage-like matrix production at the single cell level. This finding suggested that, although canonical markers have very clear functional roles in differentiation and matrix formation, their instantaneous mRNA abundance is only tenuously linked to the chondrogenic phenotype and matrix accumulation at the single cell level. One possible explanation for the apparent disconnect between gene and protein expression is that mRNA and protein exhibit different temporal dynamics. Using stochastic models of single cell behavior, we explored the impact of transcriptional stochasticity and temporal matrix dynamics on the perceived relationship between single cell mRNA and protein abundance. Simulations suggested that considering recent temporal fractions of protein (vs. total protein) increased the correlation between mRNA and protein abundance, and illustrated that mRNA stability was a crucial determinant of the timescale over which any such correlation persisted. Experimentally, non-canonical amino acid tagging was used to visualize and quantify temporal fractions of nascent extracellular matrix with high fidelity. The organization and temporal dynamics of the proteinaceous matrix depended on the biophysical features of the microenvironment, including the biomaterial scaffold and the niche constructed by cells themselves. Both chondrocytes and MSCs demonstrated marked cell-to-cell heterogeneity in nascent matrix production, consistent with model predictions. Ongoing work aims to combine these experimental measurements of nascent protein expression with instantaneous measures of mRNA abundance to better understand the mRNA-protein relationship, and to harness this understanding to improve regenerative therapies

Expression of Pea3 transcription factor genes in frontonasal and mandibular mesenchyme of the chick embryo

Fibroblast growth factors (FGFs) play an essential role in development and patterning of the vertebrate embryo. Despite extensive literature documenting the diverse roles of FGF signalling during craniofacial development, comparatively little is known about the specific downstream effectors through which FGFs influence gene expression. A previous study in our laboratory reported exogenous FGF elicited differential chondrogenic responses in frontonasal and mandibular mesenchyme (Bobick et al., 2007). Pea3 transcription factors are crucial components of the downstream effector pathway through which FGFs influence gene expression (Raible and Brand, 2001). Therefore, the purpose of my research was to examine whether differences in pea3, erm, and er81 gene expression profiles underlie the distinct responses of the frontonasal and mandibular mesenchyme cells to FGF. The present study demonstrates that FGF2 treatment differentially affects chondrogenesis in micromass cultures of frontonasal and mandibular mesenchyme isolated from stage 24/25 chick embryos. Whereas FGF2 inhibited chondrogenesis in frontonasal mesenchyme cultures, it had no effect on micromass cultures of mandibular mesenchyme. RT-qPCR and RNA dot blot analyses demonstrated that mRNA transcripts for the pea3, erm, and er81 genes are expressed by mesenchyme cells of both frontonasal and mandibular processes of stage 24/25 and stage 28/29 chick embryos. In addition, western blot data demonstrated expression of the Pea3 and Er81 proteins in micromass and explant cultures of stage 24/25 frontonasal and mandibular mesenchyme. The expression profiles of Pea3 genes were similar between the frontonasal and mandibular facial primordia prior to treatment with exogenous FGF2. However, these expression profiles were differentially altered in response to FGF2 exposure in both explant and micromass cultures. Specifically, whereas FGF2 treatment upregulated pea3 mRNA levels in explants of frontonasal mesenchyme, it had no effect on pea3 expression in mandibular explants. In micromass cultures, exogenous FGF2 elevated levels of pea3 transcripts in both frontonasal and mandibular mesenchyme. However, FGF2 treatment elevated er81 expression in frontonasal, but not mandibular mesenchyme. Conversely, exogenous FGF2 elevated erm mRNA levels in mandibular, but not frontonasal mesenchyme. Micromass cultures of mandibular mesenchyme from stage 28/29 chick embryos exhibited significantly lower levels of pea3 expression than cultures of stage 24/25 mandibular mesenchyme. This stage-dependent change correlated with a reduction in the ability of the mandibular cells to undergo spontaneous chondrogenic differentiation in micromass culture. In contrast, no stage-dependent changes in pea3 expression were observed in frontonasal mesenchyme cultures. Collectively, my data indicate that the expression profiles of pea3, erm, and er81 in frontonasal and mandibular mesenchyme become distinct only after exposure to exogenous FGF. This raises the possibility that the differences in Pea3 transcription factor expression patterns that arise in response to FGF stimuli may subsequently lead to distinct chondrogenic responses in the two facial mesenchyme populations

Spatially and Temporally Controlled Mechanical Signals to Direct Human Mesenchymal Stem Cell Behavior

In order to effectively incorporate stem cells into tissue engineering solutions, a deeper understanding of the microenvironment factors that influence their behaviors is necessary. Specifically, the inherent mechanics of the extracellular matrix (ECM) have been shown to profoundly effect multiple stem cell behaviors such as their morphology, proliferation, differentiation, and secretion of factors. The effect of matrix mechanics on stem cells has been investigated using a wide range of material systems; however, many of these systems lack the mechanical complexity that native tissues possess in terms of their spatial and temporal properties, as well as context (2D vs. 3D). In order to determine the effect of heterogeneous and dynamic mechanical signals on stem cells, a sequential crosslinking technique was developed that allowed for formation of hydrogels with a wide range in mechanical properties in terms of magnitude, context, and spatiotemporal presentation. Hydrogels with tunable mechanics were synthesized using methacrylate hyaluronic acid (MeHA) in a sequential process: 1) Michael-type `addition\u27 crosslinking using dithiothreitol to consume a fraction of the methacrylate groups, and 2) UV-initiated `radical\u27 crosslinking using controlled UV light exposure in the presence of a photoinitiator to consume unreacted methacrylates. Using this approach, we demonstrated local control of stem cell morphology, proliferation, and differentiation (adipogenesis and osteogenesis) in both patterned and gradient systems on 2D hydrogels. We further investigated the effects of mechanics in a 3D context using non-porous and porous presentations of controlled mechanics. In the non-porous system, cell behavior was shown to be dependent on mechanics as threshold responses were observed related to the ability of hMSCs to adopt a spread or rounded morphology within the hydrogel. In the 3D macroporous system, mechanics were spatially and temporally modulated and hMSC morphology, proliferation, differentiation, and secretion of angiogenic and cytokine factors were shown to be dependent on the local and temporal presentation of mechanical signals. This dissertation work emphasizes the importance of the magnitude, context, and presentation of mechanical signals and highlights this sequential crosslinking process as a model system for future investigations into heterogeneous, dynamic microenvironments, as well as a novel platform for developing future tissue engineering strategies

Effects of genetic variability on fracture healing: a temporal study of gene expression and callus phenotype

Bones have a large intrinsic capacity for repair and regeneration following an injury, however, an estimated 5-10% of nearly 8 million fractures that occur every year in the United States lead to nonunions. The process of bone regeneration is a complex trait that brings together different complements of molecular and cellular interactions to carry out its necessary mechanical functions. These interactions may be attributable to the effects of genetic variations that contribute to differences in bone morphology and fracture healing. This study was undertaken to determine how genetic variability that controls phenotypic qualities of bone affect rates and patterns of fracture healing. Three inbred strains of mice (A/J (AJ), C57BL/6J (B6), and C3H/HeJ (C3)) with known structural and biomechanical differences resulting from fetal bone development were examined. Transverse fractures were generated in the femur and healing traits were evaluated using quantitative real-time polymerase chain reaction (qRT-PCR), micro-computed tomography (μCT), biomechanical torsional testing, and cartilage contrast-enhanced micro-computed tomography (CECT). The temporal analysis of gene expression revealed that B6 had the longest duration of chondrocyte maturation and the greatest relative expression of osteogenic genes relative to either C3 or AJ. While AJ and C3 exhibited similar patterns of chondrogenesis, AJ initiated osteogenesis earlier than C3. These results suggest that compared to either AJ or B6, the C3 strain exhibited the least temporal coordination between the chondrogenic and osteogenic stages. Consistent with the relative patterns of RNA expression, μCT evaluations at day 21 post fracture showed that B6 had higher callus mineralization than AJ and C3. μCT, cartilage CECT, and biomechanical testing revealed less tissue mineralization and more cartilage near the fracture gap, which indicated a less developed bony bridge in C3 calluses at day 21 post fracture. The lack of large amounts of cartilage in calluses of all strains by day 21 indicated that all strains had initiated osteogenesis by this time. Taken together, these results showed that mice with different genetic backgrounds express different patterns of mobilization and renewal of skeletal stem cells with differing temporal progressions of chondrogenic and osteogenic differentiation. These data further show that these variations affect the phenotypic properties of fracture calluses during the process of fracture healing

Global comparative transcriptome analysis of cartilage formation in vivo

<p>Abstract</p> <p>Background</p> <p>During vertebrate embryogenesis the initial stages of bone formation by endochondral ossification involve the aggregation and proliferation of mesenchymal cells into condensations. Continued growth of the condensations and differentiation of the mesenchymal cells into chondrocytes results in the formation of cartilage templates, or anlagen, which prefigure the shape of the future bones. The chondrocytes in the anlagen further differentiate by undergoing a complex sequence of maturation and hypertrophy, and are eventually replaced by mineralized bone. Regulation of the onset of chondrogenesis is incompletely understood, and would be informed by comprehensive analyses of <it>in vivo </it>gene expression.</p> <p>Results</p> <p>Tibial and fibular pre-condensed mesenchyme was microdissected from mouse hind limbs at 11.5 dpc, and the corresponding condensations at 12.5 dpc and cartilage anlagen at 13.5 dpc. Total RNA was isolated, and cRNA generated by linear amplification was interrogated using mouse whole genome microarrays. Differential expression was validated by quantitative PCR for <it>Agc1</it>, <it>Bmp8a</it>, <it>Col2a1</it>, <it>Fgfr4</it>, <it>Foxa3</it>, <it>Gdf5</it>, <it>Klf2</it>, <it>Klf4</it>, <it>Lepre1</it>, <it>Ncad</it>, <it>Sox11</it>, and <it>Trpv4</it>. Further, independent validation of the microarray data was achieved by <it>in situ </it>hybridization to analyse the expression of <it>Lepre1</it>, <it>Pcdh8</it>, <it>Sox11</it>, and <it>Trpv4 </it>from 11.5 dpc to 13.5 dpc during mouse hind limb development. We found significant differential expression of 931 genes during these early stages of chondrogenesis. Of these, 380 genes were down-regulated and 551 up-regulated. Our studies characterized the expression pattern of gene families previously associated with chondrogenesis, such as adhesion molecules, secreted signalling molecules, transcription factors, and extracellular matrix components. Gene ontology approaches identified 892 differentially expressed genes not previously identified during the initiation of chondrogenesis. These included several <it>Bmp, Gdf, Wnt, Sox and Fox </it>family members.</p> <p>Conclusion</p> <p>These data represent the first global gene expression profiling analysis of chondrogenic tissues during <it>in vivo </it>development. They identify genes for further study on their functional roles in chondrogenesis, and provide a comprehensive and important resource for future studies on cartilage development and disease.</p

Evaluating the healing potential of PTH on femoral shaft fractures in B6, C3, and AJ mice

Parathyroid hormone is a vital mediator of bone metabolism and studies have shown that exogenous treatment can enhance the fracture repair process in murine models. Bone remodeling is a complex process that necessitates multiple molecular and cellular interactions that are affected by genetic variations. These differences contribute to both histological and whole organ level differences of fracture healing. This study was performed to determine the effect of genetic variability of fracture healing in mice treated with parathyroid hormone during two time windows. The first window was the first 14-day period post fracture associated with chondrogensis and the second was the day 15 to day 28 post fracture, which is associated with osteogenesis. Three inbred strains of mice A/J (AJ), C57BL/6J (B6), and C3H/HeJ (C3) that have material and structural differences in bone quality were given Femoral shaft fractures and healing was evaluated at different time points post fracture using quantitative real-time polymerase chain reaction (qRT-PCR) and qualitative radiographic analysis. Chondrogenic genes Sox9, ColIIa, aggrecan, and ColXa and osteogenic genes ostrix, osteocalcin, BSP, and DMP1 were examined. The temporal analysis of mRNA expression revealed that PTH treatment given in the first 14 days post fracture enhanced osteogenic and chondrogenic expression in B6 mice, but hindered expression in AJ mice. Treatment with PTH from post fracture day 14 to day 28 greatly affected the osteogenic expression of B6 mice, but had little affect on other animals. Radiographic analysis showed that each strain presents callus formation at approximately day 7 and reaches maximum size at day 21 post fracture. Additionally B6 mice appear with the largest callus and AJ the smallest. Taken together, these results are consistent with past studies in showing that different strains of mice express a unique temporal and mRNA expression pattern of chondrogenic and osteogenic differentiation. Furthermore, these variations affect the biomechanical properties of the fracture callus during bone remodeling