Gene Expression Profiling of Mouse Articular and Growth Plate Cartilage
ABSTRACT Articular cartilage is recalcitrant to repair and regeneration. Tissue engineering and regenerative medicine are potential strategies to treat the damage to articular cartilage. A thorough understanding of the gene expression profiles in articular cartilage and growth plate chondrocytes will be an important prerequisite for tissue engineering of cartilage. Regeneration is a recapitulation of embryonic development and morphogenesis. We used laser capture microdissection to capture the surface articular chondrocytes and the resting zone chondrocytes of growth plate from 14-day-old C57BL/6J mice. Total RNA was individually purified, pooled, and amplified by two rounds of in vitro transcription. Labeled cRNA probes were analyzed using the Affymetrix GeneChip Mouse Genome 430 2.0 Array. We identified 107 genes that were highly expressed by the surface articular chondrocytes and 130 genes that were highly expressed by the resting zone chondrocytes of growth plate (> or = fivefold). The expression of major matrix proteins aggrecan and collagen II were similar, while several morphogens and growth factors were differentially expressed by the surface articular chondrocytes and the resting zone chondrocytes of growth plate. The results of this investigation will be of use in the evaluation of tissue engineered cartilage.
- SourceAvailable from: Audrey McAlinden
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- "The significance of this work is further highlighted by the fact that three distinct regions of the developing limb were separated by laser capture microdissection to permit analysis of miRNA expression in precursor chondrocytes (PC), differentiated chondrocytes (DC) or hypertrophic chondrocytes (HYP) of the femur and tibia. While other studies have been carried out to identify mRNA or miRNA expression in chondrocytes from different sites of mouse or chicken cartilage tissue, either late-stage embryonic, neonatal or post-natal tissue was utilized , , . The type of study described in this report to determine miRNA expression in three distinct populations of chondrocytes at an early stage of human embryonic development (day 54–56 of gestation; prior to primary endochondral ossification) would be extremely challenging in mouse, rat or chicken tissue due to size constraints. "
ABSTRACT: There is compelling in vivo evidence from reports on human genetic mutations and transgenic mice that some microRNAs (miRNAs) play an important functional role in regulating skeletal development and growth. A number of published in vitro studies also point toward a role for miRNAs in controlling chondrocyte gene expression and differentiation. However, information on miRNAs that may regulate a specific phase of chondrocyte differentiation (i.e. production of progenitor, differentiated or hypertrophic chondrocytes) is lacking. To attempt to bridge this knowledge gap, we have investigated miRNA expression patterns in human embryonic cartilage tissue. Specifically, a developmental time point was selected, prior to endochondral ossification in the embryonic limb, to permit analysis of three distinct populations of chondrocytes. The location of chondroprogenitor cells, differentiated chondrocytes and hypertrophic chondrocytes in gestational day 54-56 human embryonic limb tissue sections was confirmed both histologically and by specific collagen expression patterns. Laser capture microdissection was utilized to separate the three chondrocyte populations and a miRNA profiling study was carried out using TaqMan® OpenArray® Human MicroRNA Panels (Applied Biosystems®). Here we report on abundantly expressed miRNAs in human embryonic cartilage tissue and, more importantly, we have identified miRNAs that are significantly differentially expressed between precursor, differentiated and hypertrophic chondrocytes by 2-fold or more. Some of the miRNAs identified in this study have been described in other aspects of cartilage or bone biology, while others have not yet been reported in chondrocytes. Finally, a bioinformatics approach was applied to begin to decipher developmental cellular pathways that may be regulated by groups of differentially expressed miRNAs during distinct stages of chondrogenesis. Data obtained from this work will serve as an important resource of information for the field of cartilage biology and will enhance our understanding of miRNA-driven mechanisms regulating cartilage and endochondral bone development, regeneration and repair.PLoS ONE 09/2013; 8(9):e75012. DOI:10.1371/journal.pone.0075012 · 3.23 Impact Factor
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- "These differences in genomic or proteomic signatures will be instrumental in defining real benchmarks for evaluating chondrogenesis and our ability to instill the desired phenotype in these cells. An important point here is to make sure that the field is using the same definitions of success; a recent microarray analysis by Reddi and co-workers comparing growth plate with articular cartilage shows profound differences between these transient and permanent cartilages, respectively (Yamane et al. 2007). Since the permanent hyaline cartilage (and the chondrocytes within) shows a remarkable ability to first establish functional matrix and then resist progressive phenotypic conversion towards the osteogenic lineage, these cells in particular should serve as our 'gold standard' for chondrogenesis. "
ABSTRACT: In this review, we outline seminal and recent work highlighting the potential of mesenchymal stem cells (MSCs) in producing cartilage-like tissue equivalents. Specific focus is placed on the mechanical properties of engineered MSC-based cartilage and how these properties relate to that of engineered cartilage based on primary chondrocytes and to native tissue properties. We discuss current limitations and/or concerns that must be addressed for the clinical realization of MSC-based cartilage therapeutics, and provide some insight into potential underpinnings for the observed deviations from chondrocyte-based engineered constructs. We posit that these differences reveal specific deficits in terms of our description of chondrogenesis, and suggest that new benchmarks must be developed towards this end. Further, we describe the growing body of literature on the mechanobiology of MSC-based cartilage, highlighting positive findings with regards to the furtherance of the chondrogenic phenotype. We likewise discuss the failure of early molecular changes to translate directly into engineered constructs with improved mechanical properties. Finally, we highlight recent work from our group and others that may point to new strategies for enhancing the formation of engineered cartilage based on MSCs.Journal of Biomechanics 10/2009; 43(1):128-36. DOI:10.1016/j.jbiomech.2009.09.018 · 2.75 Impact Factor
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- "RNA and protein analysis. GeneChip Ò Mouse Genome 430 2.0 Array (Affymetrix, Santa Clara, CA) analysis was described before . DCX was identified from a pool of genes ''Present'' in articular cartilage but ''Absent'' from the physeal cartilage. "
ABSTRACT: Articular cartilage and cartilage in the embryonic cartilaginous anlagen and growth plates are both hyaline cartilages. In this study, we found that doublecortin (DCX) was expressed in articular chondrocytes but not in chondrocytes from the cartilaginous anlagen or growth plates. DCX was expressed by the cells in the chondrogenous layers but not intermediate layer of joint interzone. Furthermore, the synovium and cruciate ligaments were DCX-negative. DCX-positive chondrocytes were very rare in tissue engineered cartilage derived from in vitro pellet culture of rat chondrosarcoma, ATDC5, and C3H10T1/2 cells. However, the new hyaline cartilage formed in rabbit knee defect contained mostly DCX-positive chondrocytes. Our results demonstrate that DCX can be used as a marker to distinguish articular chondrocytes from other chondrocytes and to evaluate the quality of tissue engineered or regenerated cartilage in terms of their "articular" or "non-articular" nature.Biochemical and Biophysical Research Communications 12/2007; 363(3):694-700. DOI:10.1016/j.bbrc.2007.09.030 · 2.28 Impact Factor